LOCUS       sequence-471550-        6596 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV zinc finger donor vector for genomic targeting.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6596)
  AUTHORS   Hockemeyer D, Soldner F, Beard C, Gao Q, Mitalipova M, Dekelver RC, 
            Katibah GE, Amora R, Boydston EA, Zeitler B, Meng X, Miller JC, 
            Zhang L, Rebar EJ, Gregory PD, Urnov FD, Jaenisch R
  TITLE     Efficient targeting of expressed and silent genes in human ESCs and 
            iPSCs using zinc-finger nucleases.
  JOURNAL   Nat Biotechnol. 2009 Sep;27(9):851-7. doi: 10.1038/nbt.1562
  PUBMED    19680244
REFERENCE   2  (bases 1 to 6596)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1038/nbt"; 
            journalName: "Nat Biotechnol"; date: "2009-09"; volume: "27"; issue:
            "9"; pages: "851-7"
COMMENT     Sequence Label: AAVS1 SA-2A-puro-pA donor
FEATURES             Location/Qualifiers
     source          1..6596
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        242..260
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    295..1098
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     misc_feature    1105..1130
                     /label=SA
                     /note="splice acceptor site"
     CDS             1154..1207
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1217..1816
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1217..1236)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1713..1733
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(1848..1865)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1854..2078
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    2098..2934
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     primer_bind     complement(2968..2987)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2969..2987)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2994..3011)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2994..3010)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3003..3025)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             3148..3450
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     3329..3348
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             3799..4593
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3799..4593
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3853..3872)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4463..4482
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             complement(join(4843..5634,5635..5703))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5466..5485
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5827..6415
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6316..6335
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6569..6586
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctca
      241 gaattaaccc tcactaaagg gactagtcct gcaggtttaa acgaattcgc cctttgcttt
      301 ctctgaccag cattctctcc cctgggcctg tgccgctttc tgtctgcagc ttgtggcctg
      361 ggtcacctct acggctggcc cagatccttc cctgccgcct ccttcaggtt ccgtcttcct
      421 ccactccctc ttccccttgc tctctgctgt gttgctgccc aaggatgctc tttccggagc
      481 acttccttct cggcgctgca ccacgtgatg tcctctgagc ggatcctccc cgtgtctggg
      541 tcctctccgg gcatctctcc tccctcaccc aaccccatgc cgtcttcact cgctgggttc
      601 ccttttcctt ctccttctgg ggcctgtgcc atctctcgtt tcttaggatg gccttctccg
      661 acggatgtct cccttgcgtc ccgcctcccc ttcttgtagg cctgcatcat caccgttttt
      721 ctggacaacc ccaaagtacc ccgtctccct ggctttagcc acctctccat cctcttgctt
      781 tctttgcctg gacaccccgt tctcctgtgg attcgggtca cctctcactc ctttcatttg
      841 ggcagctccc ctacccccct tacctctcta gtctgtgcta gctcttccag ccccctgtca
      901 tggcatcttc caggggtccg agagctcagc tagtcttctt cctccaaccc gggcccctat
      961 gtccacttca ggacagcatg tttgctgcct ccagggatcc tgtgtccccg agctgggacc
     1021 accttatatt cccagggccg gttaatgtgg ctctggttct gggtactttt atctgtcccc
     1081 tccaccccac agtggggcaa gcttctgacc tcttctcttc ctcccacagg gcctcgagag
     1141 atctggcagc ggagagggca gaggaagtct tctaacatgc ggtgacgtgg aggagaatcc
     1201 cggccctagg ctcgagatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga
     1261 cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca
     1321 caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac
     1381 gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt
     1441 ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat
     1501 ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc
     1561 gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca
     1621 gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg
     1681 ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg
     1741 cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg
     1801 caagcccggt gcctgatcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc
     1861 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     1921 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     1981 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     2041 caatagcagg catgctgggg atgcggtggg ctctatgggt cgacagtact aagctttact
     2101 agggacagga ttggtgacag aaaagcccca tccttaggcc tcctccttcc tagtctcctg
     2161 atattgggtc taacccccac ctcctgttag gcagattcct tatctggtga cacaccccca
     2221 tttcctggag ccatctctct ccttgccaga acctctaagg tttgcttacg atggagccag
     2281 agaggatcct gggagggaga gcttggcagg gggtgggagg gaaggggggg atgcgtgacc
     2341 tgcccggttc tcagtggcca ccctgcgcta ccctctccca gaacctgagc tgctctgacg
     2401 cggctgtctg gtgcgtttca ctgatcctgg tgctgcagct tccttacact tcccaagagg
     2461 agaagcagtt tggaaaaaca aaatcagaat aagttggtcc tgagttctaa ctttggctct
     2521 tcacctttct agtccccaat ttatattgtt cctccgtgcg tcagttttac ctgtgagata
     2581 aggccagtag ccagccccgt cctggcaggg ctgtggtgag gaggggggtg tccgtgtgga
     2641 aaactccctt tgtgagaatg gtgcgtccta ggtgttcacc aggtcgtggc cgcctctact
     2701 ccctttctct ttctccatcc ttctttcctt aaagagtccc cagtgctatc tgggacatat
     2761 tcctccgccc agagcagggt cccgcttccc taaggccctg ctctgggctt ctgggtttga
     2821 gtccttggca agcccaggag aggcgctcag gcttccctgt cccccttcct cgtccaccat
     2881 ctcatgcccc tggctctcct gccccttccc tacaggggtt cctggctctg ctctaagggc
     2941 gaattcgcgg ccgctaaatt caattcgccc tatagtgagt cgtattacaa ttcactggcc
     3001 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     3061 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     3121 caacagttgc gcagcctata cgtacggcag tttaaggttt acacctataa aagagagagc
     3181 cgttatcgtc tgtttgtgga tgtacagagt gatattattg acacgccggg gcgacggatg
     3241 gtgatccccc tggccagtgc acgtctgctg tcagataaag tctcccgtga actttacccg
     3301 gtggtgcata tcggggatga aagctggcgc atgatgacca ccgatatggc cagtgtgccg
     3361 gtctccgtta tcggggaaga agtggctgat ctcagccacc gcgaaaatga catcaaaaac
     3421 gccattaacc tgatgttctg gggaatataa atgtcaggca tgagattatc aaaaaggatc
     3481 ttcacctaga tccttttcac gtagaaagcc agtccgcaga aacggtgctg accccggatg
     3541 aatgtcagct actgggctat ctggacaagg gaaaacgcaa gcgcaaagag aaagcaggta
     3601 gcttgcagtg ggcttacatg gcgatagcta gactgggcgg ttttatggac agcaagcgaa
     3661 ccggaattgc cagctggggc gccctctggt aaggttggga agccctgcaa agtaaactgg
     3721 atggctttct tgccgccaag gatctgatgg cgcaggggat caagctctga tcaagagaca
     3781 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     3841 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     3901 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     3961 ggtgccctga atgaactgca agacgaggca gcgcggctat cgtggctggc cacgacgggc
     4021 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     4081 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     4141 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     4201 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     4261 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     4321 aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     4381 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     4441 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     4501 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     4561 gccttctatc gccttcttga cgagttcttc tgaattatta acgcttacaa tttcctgatg
     4621 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatcaggtg gcacttttcg
     4681 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     4741 gctcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     4801 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     4861 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     4921 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     4981 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     5041 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     5101 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     5161 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     5221 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     5281 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     5341 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgaatactc
     5401 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     5461 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     5521 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     5581 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     5641 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     5701 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgaccaa
     5761 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     5821 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     5881 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     5941 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     6001 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     6061 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     6121 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     6181 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     6241 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     6301 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     6361 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     6421 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     6481 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     6541 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaag
//
