LOCUS       sequence-399765-        5504 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  donor vector for AAVS1 targeting (puromycin selection) with multiple
            cloning site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5504)
  AUTHORS   Oceguera-Yanez F, Kim SI, Matsumoto T, Tan GW, Xiang L, Hatani T, 
            Kondo T, Ikeya M, Yoshida Y, Inoue H, Woltjen K
  TITLE     Engineering the AAVS1 locus for consistent and scalable transgene 
            expression in human iPSCs and their differentiated derivatives.
  JOURNAL   Methods. 2015 Dec 18. pii: S1046-2023(15)30181-X. doi: 
            10.1016/j.ymeth.2015.12.012.
  PUBMED    26707206
REFERENCE   2  (bases 1 to 5504)
  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"; journalName: "Methods. 
            2015 Dec 18. pii: S1046-2023(15)30181-X. doi: 
            10.1016/j.ymeth.2015.12.012."
COMMENT     Sequence Label: pAAVS1-P-MCS
FEATURES             Location/Qualifiers
     source          1..5504
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    296..825
                     /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(971..990)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(972..990)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(997..1014)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(997..1013)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1006..1028)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1155..1610
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1242..1261)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1452..1473
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1636..1740
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1741..1809,1810..2601)
                     /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     complement(1959..1978)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2772..3360
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3261..3280
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3514..3531
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3648..3669
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3684..3689,3690..3707,3708..3714)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3722..3738
                     /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     3727..3749
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3746..3762
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3746..3762
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3783..3801
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    3814..4617
                     /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    4624..4649
                     /label=SA
                     /note="splice acceptor site"
     CDS             4673..4726
                     /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             4736..5335
                     /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(4736..4755)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5232..5252
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5367..5384)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    join(5373..5504,1..93)
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
       61 gcaggcatgc tggggatgcg gtgggctcta tggactagta ttaattaaat ctagaagtcg
      121 acagtactaa gctttgacag aaaagcccca tccttaggcc tcctccttcc tagtctcctg
      181 atattgggtc taacccccac ctcctgttag gcagattcct tatctggtga cacaccccca
      241 tttcctggag ccatctctct ccttgccaga acctctaagg tttgcttacg atggagccag
      301 agaggatcct gggagggaga gcttggcagg gggtgggagg gaaggggggg atgcgtgacc
      361 tgcccggttc tcagtggcca ccctgcgcta ccctctccca gaacctgagc tgctctgacg
      421 cggctgtctg gtgcgtttca ctgatcctgg tgctgcagct tccttacact tcccaagagg
      481 agaagcagtt tggaaaaaca aaatcagaat aagttggtcc tgagttctaa ctttggctct
      541 tcacctttct agtccccaat ttatattgtt cctccgtgcg tcagttttac ctgtgagata
      601 aggccagtag ccagccccgt cctggcaggg ctgtggtgag gaggggggtg tccgtgtgga
      661 aaactccctt tgtgagaatg gtgcgtccta ggtgttcacc aggtcgtggc cgcctctact
      721 ccctttctct ttctccatcc ttctttcctt aaagagtccc cagtgctatc tgggacatat
      781 tcctccgccc agagcagggt cccgcttccc taaggccctg ctctgggctt ctgggtttga
      841 gtccttggca agcccaggag aggcgctcag gcttccctgt cccccttcct cgtccaccat
      901 ctcatgcccc tggctctcct gccccttccc tacaggggtt cctggctctg ctctagcgat
      961 cgccaattcg ccctatagtg agtcgtatta caattcactg gccgtcgttt tacaacgtcg
     1021 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     1081 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     1141 gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     1201 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     1261 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     1321 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     1381 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     1441 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     1501 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     1561 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     1621 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     1681 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     1741 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     1801 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     1861 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     1921 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     1981 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     2041 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     2101 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     2161 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     2221 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     2281 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     2341 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     2401 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     2461 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     2521 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     2581 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     2641 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     2701 accaaaatcc cttaacgtga gttttcgttc cactgagcgt caaaccccgt agaaaagatc
     2761 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     2821 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     2881 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     2941 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     3001 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     3061 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     3121 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     3181 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     3241 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     3301 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     3361 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     3421 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     3481 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     3541 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
     3601 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
     3661 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
     3721 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagctc
     3781 gaaattaacc ctcactaaag ggaacaaaag ctgtgctttc tctgaccagc attctctccc
     3841 ctgggcctgt gccgctttct gtctgcagct tgtggcctgg gtcacctcta cggctggccc
     3901 agatccttcc ctgccgcctc cttcaggttc cgtcttcctc cactccctct tccccttgct
     3961 ctctgctgtg ttgctgccca aggatgctct ttccggagca cttccttctc ggcgctgcac
     4021 cacgtgatgt cctctgagcg gatcctcccc gtgtctgggt cctctccggg catctctcct
     4081 ccctcaccca accccatgcc gtcttcactc gctgggttcc cttttccttc tccttctggg
     4141 gcctgtgcca tctctcgttt cttaggatgg ccttctccga cggatgtctc ccttgcgtcc
     4201 cgcctcccct tcttgtaggc ctgcatcatc accgtttttc tggacaaccc caaagtaccc
     4261 cgtctccctg gctttagcca cctctccatc ctcttgcttt ctttgcctgg acaccccgtt
     4321 ctcctgtgga ttcgggtcac ctctcactcc tttcatttgg gcagctcccc tacccccctt
     4381 acctctctag tctgtgctag ctcttccagc cccctgtcat ggcatcttcc aggggtccga
     4441 gagctcagct agtcttcttc ctccaacccg ggcccctatg tccacttcag gacagcatgt
     4501 ttgctgcctc cagggatcct gtgtccccga gctgggacca ccttatattc ccagggccgg
     4561 ttaatgtggc tctggttctg ggtactttta tctgtcccct ccaccccaca gtggggcaag
     4621 cttctgacct cttctcttcc tcccacaggg cctcgagaga tctggcagcg gagagggcag
     4681 aggaagtctt ctaacatgcg gtgacgtgga ggagaatccc ggccctaggc tcgagatgac
     4741 cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac
     4801 cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca
     4861 catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg
     4921 caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt
     4981 cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg
     5041 gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc
     5101 gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc
     5161 cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac
     5221 ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt
     5281 cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgatctag
     5341 agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     5401 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     5461 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtc
//
