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Vector Summary
Vector ID VB210828-1035jnw
Vector Name pLV[2CRISPR]-hCas9:T2A:Neo-U6>hMRC1[gRNA#1907]-U6>hMRC1[gRNA#5584]
Vector Size 12319 bp
Viral Genome Size 8844 bp
Vector Type Mammalian CRISPR Lentiviral Vector (Dual gRNA)
Inserted gRNA hMRC1[gRNA#1907], hMRC1[gRNA#5584]
Guide Sequence CCAGCCATGTATACGCTACT, TTGGGTTCGGGAGTCGTCGT
Inserted Nuclease hCas9:T2A:Neo
Plasmid Copy Number High
Antibiotic Resistance Ampicillin
Cloning Host VB UltraStable (or alternative strain)

Vector Map

RSV promoter
5' LTR-ΔU3
Ψ
pUC ori RRE

cPPT
AmpiciIIin U6 promoter
12000 1
hMRC1[gRNA#1907]
Terminator
U6 promoter
hMRC1[gRNA#5584]
Terminator

EFS promoter
VB210828-1035jnw 3000

SV40 early pA 9000

3' LTR-ΔU3 12319 bp

WPRE

6000

hCas9:T2A:Neo

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Vector Components
Size
Name Position Type Description Application notes
(bp)
Strong promoter; drives
Rous sarcoma virus
RSV promoter 1-229 229 Promoter transcription of viral RNA
enhancer/promoter
in packaging cells.

Truncated HIV-1 5' Allows transcription of

5' LTR-ΔU3 230-410 181 LTR long terminal viral RNA and its
repeat packaging into virus.

HIV-1 packaging Allows packaging of viral

Ψ 521-565 45 Miscellaneous
signal RNA into virus.

Rev protein binding site

that allows Rev-
HIV-1 Rev response
RRE 1075-1308 234 Miscellaneous dependent nuclear export
element
of viral RNA during viral
packaging.

Facilitates the nuclear

Central polypurine import of HIV-1 cDNA
cPPT 1803-1920 118 Miscellaneous
tract through a central DNA
flap.

Human U6 small Pol III promoter; drives

U6 promoter 1927-2175 249 Promoter
nuclear 1 promoter expression of small RNAs.

hMRC1[gRNA#1907] 2177-2272 96 gRNA None None

Allows transcription
Pol III transcription termination of small RNA
Terminator 2273-2278 6 terminator
terminator transcribed by Pol III RNA
polymerase.

Human U6 small Pol III promoter; drives

U6 promoter 2287-2535 249 Promoter expression of small RNAs.
nuclear 1 promoter

hMRC1[gRNA#5584] 2537-2632 96 gRNA None None

Allows transcription
Pol III transcription termination of small RNA
Terminator 2633-2638 6 terminator
terminator transcribed by Pol III RNA
polymerase.

Human eukaryotic
translation Medium-strength
EFS promoter 2756-2987 232 Promoter
elongation factor 1 promoter.
α1 short form

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Size
Name Position Type Description Application notes
(bp)
hCas9 and
Allows cells to express
neomycin
hCas9:T2A:Neo 3018-8144 5127 ORF hCas9 protein and be
resistance gene
resistant to neomycin.
linked by T2A

Enhances virus stability in

Woodchuck packaging cells, leading
hepatitis virus to higher titer of
WPRE 8176-8773 598 Miscellaneous
posttranscriptional packaged virus; enhances
regulatory element higher expression of
transgenes.

Allows packaging of viral

RNA into virus; self-
inactivates the 5' LTR by a
Truncated HIV-1 3'
copying mechanism
3' LTR-ΔU3 8839-9073 235 LTR long terminal
during viral genome
repeat
integration; contains
polyadenylation signal for
transcription termination.

Allows transcription
Simian virus 40 termination and
early polyadenylation of mRNA
SV40 early pA 9146-9280 135 PolyA_signal
polyadenylation transcribed by Pol II RNA
signal polymerase.

10234- AmpiciIIin Allows E. coli to be

AmpiciIIin 861 ORF
11094 resistance gene resistant to ampiciIIin.

Facilitates plasmid
replication in E. coli;
11265- pUC origin of
pUC ori 589 Rep_origin regulates high-copy
11853 replication
plasmid number (500-
700).

Note: Components added by user are listed in bold red text.

Vector Sequence
1 AATGTAGTCT TATGCAATAC TCTTGTAGTC TTGCAACATG GTAACGATGA GTTAGCAACA TGCCTTACAA GGAGAGAAAA
81 AGCACCGTGC ATGCCGATTG GTGGAAGTAA GGTGGTACGA TCGTGCCTTA TTAGGAAGGC AACAGACGGG TCTGACATGG
161 ATTGGACGAA CCACTGAATT GCCGCATTGC AGAGATATTG TATTTAAGTG CCTAGCTCGA TACATAAACG GGTCTCTCTG
241 GTTAGACCAG ATCTGAGCCT GGGAGCTCTC TGGCTAACTA GGGAACCCAC TGCTTAAGCC TCAATAAAGC TTGCCTTGAG
321 TGCTTCAAGT AGTGTGTGCC CGTCTGTTGT GTGACTCTGG TAACTAGAGA TCCCTCAGAC CCTTTTAGTC AGTGTGGAAA
401 ATCTCTAGCA GTGGCGCCCG AACAGGGACT TGAAAGCGAA AGGGAAACCA GAGGAGCTCT CTCGACGCAG GACTCGGCTT
481 GCTGAAGCGC GCACGGCAAG AGGCGAGGGG CGGCGACTGG TGAGTACGCC AAAAATTTTG ACTAGCGGAG GCTAGAAGGA
561 GAGAGATGGG TGCGAGAGCG TCAGTATTAA GCGGGGGAGA ATTAGATCGC GATGGGAAAA AATTCGGTTA AGGCCAGGGG

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641 GAAAGAAAAA ATATAAATTA AAACATATAG TATGGGCAAG CAGGGAGCTA GAACGATTCG CAGTTAATCC TGGCCTGTTA
721 GAAACATCAG AAGGCTGTAG ACAAATACTG GGACAGCTAC AACCATCCCT TCAGACAGGA TCAGAAGAAC TTAGATCATT
801 ATATAATACA GTAGCAACCC TCTATTGTGT GCATCAAAGG ATAGAGATAA AAGACACCAA GGAAGCTTTA GACAAGATAG
881 AGGAAGAGCA AAACAAAAGT AAGACCACCG CACAGCAAGC GGCCGCTGAT CTTCAGACCT GGAGGAGGAG ATATGAGGGA
961 CAATTGGAGA AGTGAATTAT ATAAATATAA AGTAGTAAAA ATTGAACCAT TAGGAGTAGC ACCCACCAAG GCAAAGAGAA
1041 GAGTGGTGCA GAGAGAAAAA AGAGCAGTGG GAATAGGAGC TTTGTTCCTT GGGTTCTTGG GAGCAGCAGG AAGCACTATG
1121 GGCGCAGCGT CAATGACGCT GACGGTACAG GCCAGACAAT TATTGTCTGG TATAGTGCAG CAGCAGAACA ATTTGCTGAG
1201 GGCTATTGAG GCGCAACAGC ATCTGTTGCA ACTCACAGTC TGGGGCATCA AGCAGCTCCA GGCAAGAATC CTGGCTGTGG
1281 AAAGATACCT AAAGGATCAA CAGCTCCTGG GGATTTGGGG TTGCTCTGGA AAACTCATTT GCACCACTGC TGTGCCTTGG
1361 AATGCTAGTT GGAGTAATAA ATCTCTGGAA CAGATTTGGA ATCACACGAC CTGGATGGAG TGGGACAGAG AAATTAACAA
1441 TTACACAAGC TTAATACACT CCTTAATTGA AGAATCGCAA AACCAGCAAG AAAAGAATGA ACAAGAATTA TTGGAATTAG
1521 ATAAATGGGC AAGTTTGTGG AATTGGTTTA ACATAACAAA TTGGCTGTGG TATATAAAAT TATTCATAAT GATAGTAGGA
1601 GGCTTGGTAG GTTTAAGAAT AGTTTTTGCT GTACTTTCTA TAGTGAATAG AGTTAGGCAG GGATATTCAC CATTATCGTT
1681 TCAGACCCAC CTCCCAACCC CGAGGGGACC CGACAGGCCC GAAGGAATAG AAGAAGAAGG TGGAGAGAGA GACAGAGACA
1761 GATCCATTCG ATTAGTGAAC GGATCTCGAC GGTATCGCTA GCTTTTAAAA GAAAAGGGGG GATTGGGGGG TACAGTGCAG
1841 GGGAAAGAAT AGTAGACATA ATAGCAACAG ACATACAAAC TAAAGAATTA CAAAAACAAA TTACAAAAAT TCAAAATTTT
1921 ACTAGTGAGG GCCTATTTCC CATGATTCCT TCATATTTGC ATATACGATA CAAGGCTGTT AGAGAGATAA TTGGAATTAA
2001 TTTGACTGTA AACACAAAGA TATTAGTACA AAATACGTGA CGTAGAAAGT AATAATTTCT TGGGTAGTTT GCAGTTTTAA
2081 AATTATGTTT TAAAATGGAC TATCATATGC TTACCGTAAC TTGAAAGTAT TTCGATTTCT TGGCTTTATA TATCTTGTGG
2161 AAAGGACGAA ACACCGCCAG CCATGTATAC GCTACTGTTT TAGAGCTAGA AATAGCAAGT TAAAATAAGG CTAGTCCGTT
2241 ATCAACTTGA AAAAGTGGCA CCGAGTCGGT GCTTTTTTGG CCGGCCGAGG GCCTATTTCC CATGATTCCT TCATATTTGC
2321 ATATACGATA CAAGGCTGTT AGAGAGATAA TTGGAATTAA TTTGACTGTA AACACAAAGA TATTAGTACA AAATACGTGA
2401 CGTAGAAAGT AATAATTTCT TGGGTAGTTT GCAGTTTTAA AATTATGTTT TAAAATGGAC TATCATATGC TTACCGTAAC
2481 TTGAAAGTAT TTCGATTTCT TGGCTTTATA TATCTTGTGG AAAGGACGAA ACACCGTTGG GTTCGGGAGT CGTCGTGTTT
2561 TAGAGCTAGA AATAGCAAGT TAAAATAAGG CTAGTCCGTT ATCAACTTGA AAAAGTGGCA CCGAGTCGGT GCTTTTTTGT
2641 TTTAGAGCTA GAAATAGCAA GTTAAAATAA GGCTAGTCCG TTTTTAGCGC GTGCGCCAAT TCTGCAGACA AATGGCTCTA
2721 GAGGTACCGA ATTCCAACTT TGTATAGAAA AGTTGGGCTC CGGTGCCCGT CAGTGGGCAG AGCGCACATC GCCCACAGTC
2801 CCCGAGAAGT TGGGGGGAGG GGTCGGCAAT TGATCCGGTG CCTAGAGAAG GTGGCGCGGG GTAAACTGGG AAAGTGATGT
2881 CGTGTACTGG CTCCGCCTTT TTCCCGAGGG TGGGGGAGAA CCGTATATAA GTGCAGTAGT CGCCGTGAAC GTTCTTTTTC
2961 GCAACGGGTT TGCCGCCAGA ACACAGGCAA GTTTGTACAA AAAAGCAGGC TGCCACCATG GACTATAAGG ACCACGACGG
3041 AGACTACAAG GATCATGATA TTGATTACAA AGACGATGAC GATAAGATGG CCCCAAAGAA GAAGCGGAAG GTCGGTATCC
3121 ACGGAGTCCC AGCAGCCGAC AAGAAGTACA GCATCGGCCT GGACATCGGC ACCAACTCTG TGGGCTGGGC CGTGATCACC
3201 GACGAGTACA AGGTGCCCAG CAAGAAATTC AAGGTGCTGG GCAACACCGA CCGGCACAGC ATCAAGAAGA ACCTGATCGG
3281 AGCCCTGCTG TTCGACAGCG GCGAAACAGC CGAGGCCACC CGGCTGAAGA GAACCGCCAG AAGAAGATAC ACCAGACGGA
3361 AGAACCGGAT CTGCTATCTG CAAGAGATCT TCAGCAACGA GATGGCCAAG GTGGACGACA GCTTCTTCCA CAGACTGGAA
3441 GAGTCCTTCC TGGTGGAAGA GGATAAGAAG CACGAGCGGC ACCCCATCTT CGGCAACATC GTGGACGAGG TGGCCTACCA
3521 CGAGAAGTAC CCCACCATCT ACCACCTGAG AAAGAAACTG GTGGACAGCA CCGACAAGGC CGACCTGCGG CTGATCTATC
3601 TGGCCCTGGC CCACATGATC AAGTTCCGGG GCCACTTCCT GATCGAGGGC GACCTGAACC CCGACAACAG CGACGTGGAC
3681 AAGCTGTTCA TCCAGCTGGT GCAGACCTAC AACCAGCTGT TCGAGGAAAA CCCCATCAAC GCCAGCGGCG TGGACGCCAA
3761 GGCCATCCTG TCTGCCAGAC TGAGCAAGAG CAGACGGCTG GAAAATCTGA TCGCCCAGCT GCCCGGCGAG AAGAAGAATG
3841 GCCTGTTCGG AAACCTGATT GCCCTGAGCC TGGGCCTGAC CCCCAACTTC AAGAGCAACT TCGACCTGGC CGAGGATGCC
3921 AAACTGCAGC TGAGCAAGGA CACCTACGAC GACGACCTGG ACAACCTGCT GGCCCAGATC GGCGACCAGT ACGCCGACCT
4001 GTTTCTGGCC GCCAAGAACC TGTCCGACGC CATCCTGCTG AGCGACATCC TGAGAGTGAA CACCGAGATC ACCAAGGCCC
4081 CCCTGAGCGC CTCTATGATC AAGAGATACG ACGAGCACCA CCAGGACCTG ACCCTGCTGA AAGCTCTCGT GCGGCAGCAG
4161 CTGCCTGAGA AGTACAAAGA GATTTTCTTC GACCAGAGCA AGAACGGCTA CGCCGGCTAC ATTGACGGCG GAGCCAGCCA
4241 GGAAGAGTTC TACAAGTTCA TCAAGCCCAT CCTGGAAAAG ATGGACGGCA CCGAGGAACT GCTCGTGAAG CTGAACAGAG
4321 AGGACCTGCT GCGGAAGCAG CGGACCTTCG ACAACGGCAG CATCCCCCAC CAGATCCACC TGGGAGAGCT GCACGCCATT
4401 CTGCGGCGGC AGGAAGATTT TTACCCATTC CTGAAGGACA ACCGGGAAAA GATCGAGAAG ATCCTGACCT TCCGCATCCC

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4481 CTACTACGTG GGCCCTCTGG CCAGGGGAAA CAGCAGATTC GCCTGGATGA CCAGAAAGAG CGAGGAAACC ATCACCCCCT

4561 GGAACTTCGA GGAAGTGGTG GACAAGGGCG CTTCCGCCCA GAGCTTCATC GAGCGGATGA CCAACTTCGA TAAGAACCTG
4641 CCCAACGAGA AGGTGCTGCC CAAGCACAGC CTGCTGTACG AGTACTTCAC CGTGTATAAC GAGCTGACCA AAGTGAAATA
4721 CGTGACCGAG GGAATGAGAA AGCCCGCCTT CCTGAGCGGC GAGCAGAAAA AGGCCATCGT GGACCTGCTG TTCAAGACCA
4801 ACCGGAAAGT GACCGTGAAG CAGCTGAAAG AGGACTACTT CAAGAAAATC GAGTGCTTCG ACTCCGTGGA AATCTCCGGC
4881 GTGGAAGATC GGTTCAACGC CTCCCTGGGC ACATACCACG ATCTGCTGAA AATTATCAAG GACAAGGACT TCCTGGACAA
4961 TGAGGAAAAC GAGGACATTC TGGAAGATAT CGTGCTGACC CTGACACTGT TTGAGGACAG AGAGATGATC GAGGAACGGC
5041 TGAAAACCTA TGCCCACCTG TTCGACGACA AAGTGATGAA GCAGCTGAAG CGGCGGAGAT ACACCGGCTG GGGCAGGCTG
5121 AGCCGGAAGC TGATCAACGG CATCCGGGAC AAGCAGTCCG GCAAGACAAT CCTGGATTTC CTGAAGTCCG ACGGCTTCGC
5201 CAACAGAAAC TTCATGCAGC TGATCCACGA CGACAGCCTG ACCTTTAAAG AGGACATCCA GAAAGCCCAG GTGTCCGGCC
5281 AGGGCGATAG CCTGCACGAG CACATTGCCA ATCTGGCCGG CAGCCCCGCC ATTAAGAAGG GCATCCTGCA GACAGTGAAG
5361 GTGGTGGACG AGCTCGTGAA AGTGATGGGC CGGCACAAGC CCGAGAACAT CGTGATCGAA ATGGCCAGAG AGAACCAGAC
5441 CACCCAGAAG GGACAGAAGA ACAGCCGCGA GAGAATGAAG CGGATCGAAG AGGGCATCAA AGAGCTGGGC AGCCAGATCC
5521 TGAAAGAACA CCCCGTGGAA AACACCCAGC TGCAGAACGA GAAGCTGTAC CTGTACTACC TGCAGAATGG GCGGGATATG
5601 TACGTGGACC AGGAACTGGA CATCAACCGG CTGTCCGACT ACGATGTGGA CCATATCGTG CCTCAGAGCT TTCTGAAGGA
5681 CGACTCCATC GACAACAAGG TGCTGACCAG AAGCGACAAG AACCGGGGCA AGAGCGACAA CGTGCCCTCC GAAGAGGTCG
5761 TGAAGAAGAT GAAGAACTAC TGGCGGCAGC TGCTGAACGC CAAGCTGATT ACCCAGAGAA AGTTCGACAA TCTGACCAAG
5841 GCCGAGAGAG GCGGCCTGAG CGAACTGGAT AAGGCCGGCT TCATCAAGAG ACAGCTGGTG GAAACCCGGC AGATCACAAA
5921 GCACGTGGCA CAGATCCTGG ACTCCCGGAT GAACACTAAG TACGACGAGA ATGACAAGCT GATCCGGGAA GTGAAAGTGA
6001 TCACCCTGAA GTCCAAGCTG GTGTCCGATT TCCGGAAGGA TTTCCAGTTT TACAAAGTGC GCGAGATCAA CAACTACCAC
6081 CACGCCCACG ACGCCTACCT GAACGCCGTC GTGGGAACCG CCCTGATCAA AAAGTACCCT AAGCTGGAAA GCGAGTTCGT
6161 GTACGGCGAC TACAAGGTGT ACGACGTGCG GAAGATGATC GCCAAGAGCG AGCAGGAAAT CGGCAAGGCT ACCGCCAAGT
6241 ACTTCTTCTA CAGCAACATC ATGAACTTTT TCAAGACCGA GATTACCCTG GCCAACGGCG AGATCCGGAA GCGGCCTCTG
6321 ATCGAGACAA ACGGCGAAAC CGGGGAGATC GTGTGGGATA AGGGCCGGGA TTTTGCCACC GTGCGGAAAG TGCTGAGCAT
6401 GCCCCAAGTG AATATCGTGA AAAAGACCGA GGTGCAGACA GGCGGCTTCA GCAAAGAGTC TATCCTGCCC AAGAGGAACA
6481 GCGATAAGCT GATCGCCAGA AAGAAGGACT GGGACCCTAA GAAGTACGGC GGCTTCGACA GCCCCACCGT GGCCTATTCT
6561 GTGCTGGTGG TGGCCAAAGT GGAAAAGGGC AAGTCCAAGA AACTGAAGAG TGTGAAAGAG CTGCTGGGGA TCACCATCAT
6641 GGAAAGAAGC AGCTTCGAGA AGAATCCCAT CGACTTTCTG GAAGCCAAGG GCTACAAAGA AGTGAAAAAG GACCTGATCA
6721 TCAAGCTGCC TAAGTACTCC CTGTTCGAGC TGGAAAACGG CCGGAAGAGA ATGCTGGCCT CTGCCGGCGA ACTGCAGAAG
6801 GGAAACGAAC TGGCCCTGCC CTCCAAATAT GTGAACTTCC TGTACCTGGC CAGCCACTAT GAGAAGCTGA AGGGCTCCCC
6881 CGAGGATAAT GAGCAGAAAC AGCTGTTTGT GGAACAGCAC AAGCACTACC TGGACGAGAT CATCGAGCAG ATCAGCGAGT
6961 TCTCCAAGAG AGTGATCCTG GCCGACGCTA ATCTGGACAA AGTGCTGTCC GCCTACAACA AGCACCGGGA TAAGCCCATC
7041 AGAGAGCAGG CCGAGAATAT CATCCACCTG TTTACCCTGA CCAATCTGGG AGCCCCTGCC GCCTTCAAGT ACTTTGACAC
7121 CACCATCGAC CGGAAGAGGT ACACCAGCAC CAAAGAGGTG CTGGACGCCA CCCTGATCCA CCAGAGCATC ACCGGCCTGT
7201 ACGAGACACG GATCGACCTG TCTCAGCTGG GAGGCGACAA AAGGCCGGCG GCCACGAAAA AGGCCGGCCA GGCAAAAAAG
7281 AAAAAGGGCT CCGGAGAGGG CAGGGGAAGT CTTCTAACAT GCGGGGACGT GGAGGAAAAT CCCGGCCCCA TGATTGAACA
7361 AGATGGATTG CACGCAGGTT CTCCGGCCGC TTGGGTGGAG AGGCTATTCG GCTATGACTG GGCACAACAG ACAATCGGCT
7441 GCTCTGATGC CGCCGTGTTC CGGCTGTCAG CGCAGGGGCG CCCGGTTCTT TTTGTCAAGA CCGACCTGTC CGGTGCCCTG
7521 AATGAACTGC AAGACGAGGC AGCGCGGCTA TCGTGGCTGG CCACGACGGG CGTTCCTTGC GCAGCTGTGC TCGACGTTGT
7601 CACTGAAGCG GGAAGGGACT GGCTGCTATT GGGCGAAGTG CCGGGGCAGG ATCTCCTGTC ATCTCACCTT GCTCCTGCCG
7681 AGAAAGTATC CATCATGGCT GATGCAATGC GGCGGCTGCA TACGCTTGAT CCGGCTACCT GCCCATTCGA CCACCAAGCG
7761 AAACATCGCA TCGAGCGAGC ACGTACTCGG ATGGAAGCCG GTCTTGTCGA TCAGGATGAT CTGGACGAAG AGCATCAGGG
7841 GCTCGCGCCA GCCGAACTGT TCGCCAGGCT CAAGGCGAGC ATGCCCGACG GCGAGGATCT CGTCGTGACC CATGGCGATG
7921 CCTGCTTGCC GAATATCATG GTGGAAAATG GCCGCTTTTC TGGATTCATC GACTGTGGCC GGCTGGGTGT GGCGGACCGC
8001 TATCAGGACA TAGCGTTGGC TACCCGTGAT ATTGCTGAAG AGCTTGGCGG CGAATGGGCT GACCGCTTCC TCGTGCTTTA
8081 CGGTATCGCC GCTCCCGATT CGCAGCGCAT CGCCTTCTAT CGCCTTCTTG ACGAGTTCTT CTGAACCCAG CTTTCTTGTA
8161 CAAAGTGGTG GTACCCGATA ATCAACCTCT GGATTACAAA ATTTGTGAAA GATTGACTGG TATTCTTAAC TATGTTGCTC
CTTTTACGCT ATGTGGATAC GCTGCTTTAA TGCCTTTGTA TCATGCTATT GCTTCCCGTA TGGCTTTCAT TTTCTCCTCC

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8241 TTGTATAAAT CCTGGTTGCT GTCTCTTTAT GAGGAGTTGT GGCCCGTTGT CAGGCAACGT GGCGTGGTGT GCACTGTGTT
8321 TGCTGACGCA ACCCCCACTG GTTGGGGCAT TGCCACCACC TGTCAGCTCC TTTCCGGGAC TTTCGCTTTC CCCCTCCCTA
8401 TTGCCACGGC GGAACTCATC GCCGCCTGCC TTGCCCGCTG CTGGACAGGG GCTCGGCTGT TGGGCACTGA CAATTCCGTG
8481 GTGTTGTCGG GGAAGCTGAC GTCCTTTCCA TGGCTGCTCG CCTGTGTTGC CACCTGGATT CTGCGCGGGA CGTCCTTCTG
8561 CTACGTCCCT TCGGCCCTCA ATCCAGCGGA CCTTCCTTCC CGCGGCCTGC TGCCGGCTCT GCGGCCTCTT CCGCGTCTTC
8641 GCCTTCGCCC TCAGACGAGT CGGATCTCCC TTTGGGCCGC CTCCCCGCAT CGGCTTTAAG ACCAATGACT TACAAGGCAG
8721 CTGTAGATCT TAGCCACTTT TTAAAAGAAA AGGGGGGACT GGAAGGGCTA ATTCACTCCC AACGAAGACA AGATCTGCTT
8801 TTTGCTTGTA CTGGGTCTCT CTGGTTAGAC CAGATCTGAG CCTGGGAGCT CTCTGGCTAA CTAGGGAACC CACTGCTTAA
8881 GCCTCAATAA AGCTTGCCTT GAGTGCTTCA AGTAGTGTGT GCCCGTCTGT TGTGTGACTC TGGTAACTAG AGATCCCTCA
8961 GACCCTTTTA GTCAGTGTGG AAAATCTCTA GCAGTAGTAG TTCATGTCAT CTTATTATTC AGTATTTATA ACTTGCAAAG
9041 AAATGAATAT CAGAGAGTGA GAGGAACTTG TTTATTGCAG CTTATAATGG TTACAAATAA AGCAATAGCA TCACAAATTT
9121 CACAAATAAA GCATTTTTTT CACTGCATTC TAGTTGTGGT TTGTCCAAAC TCATCAATGT ATCTTATCAT GTCTGGCTCT
9201 AGCTATCCCG CCCCTAACTC CGCCCATCCC GCCCCTAACT CCGCCCAGTT CCGCCCATTC TCCGCCCCAT GGCTGACTAA
9281 TTTTTTTTAT TTATGCAGAG GCCGAGGCCG CCTCGGCCTC TGAGCTATTC CAGAAGTAGT GAGGAGGCTT TTTTGGAGGC
9361 CTAGGGACGT ACCCAATTCG CCCTATAGTG AGTCGTATTA CGCGCGCTCA CTGGCCGTCG TTTTACAACG TCGTGACTGG
9441 GAAAACCCTG GCGTTACCCA ACTTAATCGC CTTGCAGCAC ATCCCCCTTT CGCCAGCTGG CGTAATAGCG AAGAGGCCCG
9521 CACCGATCGC CCTTCCCAAC AGTTGCGCAG CCTGAATGGC GAATGGGACG CGCCCTGTAG CGGCGCATTA AGCGCGGCGG
9601 GTGTGGTGGT TACGCGCAGC GTGACCGCTA CACTTGCCAG CGCCCTAGCG CCCGCTCCTT TCGCTTTCTT CCCTTCCTTT
9681 CTCGCCACGT TCGCCGGCTT TCCCCGTCAA GCTCTAAATC GGGGGCTCCC TTTAGGGTTC CGATTTAGTG CTTTACGGCA
9761 CCTCGACCCC AAAAAACTTG ATTAGGGTGA TGGTTCACGT AGTGGGCCAT CGCCCTGATA GACGGTTTTT CGCCCTTTGA
9841 CGTTGGAGTC CACGTTCTTT AATAGTGGAC TCTTGTTCCA AACTGGAACA ACACTCAACC CTATCTCGGT CTATTCTTTT
9921 GATTTATAAG GGATTTTGCC GATTTCGGCC TATTGGTTAA AAAATGAGCT GATTTAACAA AAATTTAACG CGAATTTTAA
10001 CAAAATATTA ACGCTTACAA TTTAGGTGGC ACTTTTCGGG GAAATGTGCG CGGAACCCCT ATTTGTTTAT TTTTCTAAAT
10081 ACATTCAAAT ATGTATCCGC TCATGAGACA ATAACCCTGA TAAATGCTTC AATAATATTG AAAAAGGAAG AGTATGAGTA
10161 TTCAACATTT CCGTGTCGCC CTTATTCCCT TTTTTGCGGC ATTTTGCCTT CCTGTTTTTG CTCACCCAGA AACGCTGGTG
10241 AAAGTAAAAG ATGCTGAAGA TCAGTTGGGT GCACGAGTGG GTTACATCGA ACTGGATCTC AACAGCGGTA AGATCCTTGA
10321 GAGTTTTCGC CCCGAAGAAC GTTTTCCAAT GATGAGCACT TTTAAAGTTC TGCTATGTGG CGCGGTATTA TCCCGTATTG
10401 ACGCCGGGCA AGAGCAACTC GGTCGCCGCA TACACTATTC TCAGAATGAC TTGGTTGAGT ACTCACCAGT CACAGAAAAG
10481 CATCTTACGG ATGGCATGAC AGTAAGAGAA TTATGCAGTG CTGCCATAAC CATGAGTGAT AACACTGCGG CCAACTTACT
10561 TCTGACAACG ATCGGAGGAC CGAAGGAGCT AACCGCTTTT TTGCACAACA TGGGGGATCA TGTAACTCGC CTTGATCGTT
10641 GGGAACCGGA GCTGAATGAA GCCATACCAA ACGACGAGCG TGACACCACG ATGCCTGTAG CAATGGCAAC AACGTTGCGC
10721 AAACTATTAA CTGGCGAACT ACTTACTCTA GCTTCCCGGC AACAATTAAT AGACTGGATG GAGGCGGATA AAGTTGCAGG
10801 ACCACTTCTG CGCTCGGCCC TTCCGGCTGG CTGGTTTATT GCTGATAAAT CTGGAGCCGG TGAGCGTGGG TCTCGCGGTA
10881 TCATTGCAGC ACTGGGGCCA GATGGTAAGC CCTCCCGTAT CGTAGTTATC TACACGACGG GGAGTCAGGC AACTATGGAT
10961 GAACGAAATA GACAGATCGC TGAGATAGGT GCCTCACTGA TTAAGCATTG GTAACTGTCA GACCAAGTTT ACTCATATAT
11041 ACTTTAGATT GATTTAAAAC TTCATTTTTA ATTTAAAAGG ATCTAGGTGA AGATCCTTTT TGATAATCTC ATGACCAAAA
11121 TCCCTTAACG TGAGTTTTCG TTCCACTGAG CGTCAGACCC CGTAGAAAAG ATCAAAGGAT CTTCTTGAGA TCCTTTTTTT
11201 CTGCGCGTAA TCTGCTGCTT GCAAACAAAA AAACCACCGC TACCAGCGGT GGTTTGTTTG CCGGATCAAG AGCTACCAAC
11281 TCTTTTTCCG AAGGTAACTG GCTTCAGCAG AGCGCAGATA CCAAATACTG TTCTTCTAGT GTAGCCGTAG TTAGGCCACC
11361 ACTTCAAGAA CTCTGTAGCA CCGCCTACAT ACCTCGCTCT GCTAATCCTG TTACCAGTGG CTGCTGCCAG TGGCGATAAG
11441 TCGTGTCTTA CCGGGTTGGA CTCAAGACGA TAGTTACCGG ATAAGGCGCA GCGGTCGGGC TGAACGGGGG GTTCGTGCAC
11521 ACAGCCCAGC TTGGAGCGAA CGACCTACAC CGAACTGAGA TACCTACAGC GTGAGCTATG AGAAAGCGCC ACGCTTCCCG
11601 AAGAGAGAAA GGCGGACAGG TATCCGGTAA GCGGCAGGGT CGGAACAGGA GAGCGCACGA GGGAGCTTCC AGGGGGAAAC
11681 GCCTGGTATC TTTATAGTCC TGTCGGGTTT CGCCACCTCT GACTTGAGCG TCGATTTTTG TGATGCTCGT CAGGGGGGCG
11761 GAGCCTATGG AAAAACGCCA GCAACGCGGC CTTTTTACGG TTCCTGGCCT TTTGCTGGCC TTTTGCTCAC ATGTTCTTTC
11841 CTGCGTTATC CCCTGATTCT GTGGATAACC GTATTACCGC CTTTGAGTGA GCTGATACCG CTCGCCGCAG CCGAACGACC
11921 GAGCGCAGCG AGTCAGTGAG CGAGGAAGCG GAAGAGCGCC CAATACGCAA ACCGCCTCTC CCCGCGCGTT GGCCGATTCA
12001 TTAATGCAGC TGGCACGACA GGTTTCCCGA CTGGAAAGCG GGCAGTGAGC GCAACGCAAT TAATGTGAGT TAGCTCACTC

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12081 ATTAGGCACC CCAGGCTTTA CACTTTATGC TTCCGGCTCG TATGTTGTGT GGAATTGTGA GCGGATAACA ATTTCACACA

12161 GGAAACAGCT ATGACCATGA TTACGCCAAG CGCGCAATTA ACCCTCACTA AAGGGAACAA AAGCTGGAGC TGCAAGCTT
12241

Validation by Restriction Enzyme Digestion

Restriction Enzymes Cutting Sites DNA Fragments (bp)
ApaLI 8390, 10350, 11596 1960, 1246, 9113
SacII 8684 12319
ApaLI+SacII 8390, 8684, 10350, 11596 294, 1666, 1246, 9113

Vector # : VB210828-1035jnw Page 7 / 7