LOCUS       sequence-138857-        6909 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6909)
  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 6909)
  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: TV-hPITX3-forward
FEATURES             Location/Qualifiers
     source          1..6909
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(11..28)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(182..770)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(262..281)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(941..1732,1733..1801))
                     /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     1564..1583
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1802..1906)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1974..1992
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2030..2052)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2152..2171
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2252..2270
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2252..2269
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      3124..3133
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3130..3132,3133..3135,3136..3849)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3130..3132,3133..3135,3136..3846)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3175..3196)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3436..3455)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3783..3804
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3972..4093
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4009..4028)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4063..4082
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    4137..4170
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4231..4730
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4266..4289)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4663..4682
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             4770..5369
                     /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(4770..4789)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5266..5286
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    5926..5959
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(6814..6833)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6815..6833)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
       61 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
      121 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
      181 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      241 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      301 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      361 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      421 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      481 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      541 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      601 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
      661 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      721 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      781 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
      841 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
      901 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
      961 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     1021 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     1081 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     1141 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     1201 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     1261 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     1321 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     1381 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     1441 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     1501 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     1561 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     1621 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     1681 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     1741 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     1801 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     1861 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     1921 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     1981 gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     2041 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     2101 gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag
     2161 agcagattgt actgagagtg caccatatgg acatattgtc gttagaacgc ggctacaatt
     2221 aatacataac cttatgtatc atacacatac gatttaggtg acactataga actcgagcag
     2281 gtatgattgg ctgaggacca tgtatgagta tagggggata ctaagcattc aggtgagaag
     2341 gattttaagt ttttgcagat gtagtatagg gtatgtagcg tgctatagtg tagattttgt
     2401 tgttgttgtt ttttgagaca gcatcttgct ctgtcaccca ggctggagtg cagtagcacc
     2461 atctcggctc gctgcaacct ccgcctccca ggttcaagcg attctcctgc ctcagcctcc
     2521 cgagtagctg agattacagg cacgtgccac cccgcccagc aatttttgta tttttagtag
     2581 ggacgggatt ttaccatgtt ggccaggctg gtctcgaact cctgacctca ggtgatccac
     2641 ccgccttggc ctcccaaagt gctgggatta caggcgtgag ccactgcgcc cggcctatag
     2701 tctacatttt tattaattca ataaatattg aaccccaact gtatacctgg cacagctcta
     2761 gttcccagca gtaaacgaaa cgaagtctgg tttatatatc tatgcagaga acctctcagc
     2821 atgcacctct gcgtccttag gatgctattt cccgattcta tcctaaggag ccccactcag
     2881 ccccagcttt acggctgggg ttgagaaagg cgccagggaa tttacgagga aacgctgctg
     2941 ggacctagct aagccggaga ttttaccccc attaccctgg tctgtgtctt ctctcgcctc
     3001 cagagccctc cctccatgga gttcggcctg cccagcgagg cagaggcccg gagccctgcc
     3061 ctgtcgctgt cagacgctgg cactccgcac ccccagctcc cagagcacgg ggatccaccg
     3121 gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     3181 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
     3241 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     3301 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
     3361 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     3421 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     3481 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     3541 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
     3601 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
     3661 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     3721 gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat
     3781 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     3841 tacaagtaaa gcggccgcga ctctagatca taatcagcca taccacattt gtagaggttt
     3901 tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa
     3961 ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     4021 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     4081 tcaatgtatc ttaaggtacc gagctcgaat tccgatcata ttcaataacc cttaatataa
     4141 cttcgtataa tgtatgctat acgaagttat taggtctgaa gaggagttta cgtccagcca
     4201 agcttaggat ctcgacctcg aaattctacc gggtagggga ggcgcttttc ccaaggcagt
     4261 ctggagcatg cgctttagca gccccgctgg gcacttggcg ctacacaagt ggcctctggc
     4321 ctcgcacaca ttccacatcc accggtaggc gccaaccggc tccgttcttt ggtggcccct
     4381 tcgcgccacc ttctactcct cccctagtca ggaagttccc ccccgccccg cagctcgcgt
     4441 cgtgcaggac gtgacaaatg gaagtagcac gtctcactag tctcgtgcag atggacagca
     4501 ccgctgagca atggaagcgg gtaggccttt ggggcagcgg ccaatagcag ctttgctcct
     4561 tcgctttctg ggctcagagg ctgggaaggg gtgggtccgg gggcgggctc aggggcgggc
     4621 tcaggggcgg ggcgggcgcc cgaaggtcct ccggaggccc ggcattctgc acgcttcaaa
     4681 agcgcacgtc tgccgcgctg ttctcctctt cctcatctcc gggcctttcg acctgcatcc
     4741 atctagatct cgagcagctg aagcttacca tgaccgagta caagcccacg gtgcgcctcg
     4801 ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc
     4861 ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag
     4921 aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg
     4981 ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga
     5041 tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag
     5101 gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct
     5161 cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg
     5221 ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct
     5281 acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct
     5341 ggtgcatgac ccgcaagccc ggtgcctgac gcccgcccca cgacccgcag cgcccgaccg
     5401 aaaggagcgc acgaccccat gcatcgatga tatcagatcc ccgggatgca gaaattgatg
     5461 atctattaaa caataaagat gtccactaaa atggaagttt ttcctgtcat actttgttaa
     5521 gaagggtgag aacagagtac ctacattttg aatggaagga ttggagctac gggggtgggg
     5581 gtggggtggg attagataaa tgcctgctct ttactgaagg ctctttacta ttgctttatg
     5641 ataatgtttc atagttggat atcataattt aaacaagcaa aaccaaatta agggccagct
     5701 cattcctccc actcatgatc tatagatcta tagatctctc gtgggatcat tgtttttctc
     5761 ttgattccca ctttgtggtt ctaagtactg tggtttccaa atgtgtcagt ttcatagcct
     5821 gaagaacgag atcagcagcc tctgttccac atacacttca ttctcagtat tgttttgcca
     5881 agttctaatt ccatcagaag ctggtcgaga tccggaaccc ttaatataac ttcgtataat
     5941 gtatgctata cgaagttatt aggtccctcg aagaggttca ctagtactgg ccattgcggc
     6001 catcctgcaa gggccaggag cacagcggta agcgcgcccc cttccggggg tgcaggacat
     6061 aacagcttca tccccagtgc gggtgggacc ccggctgggc agccccggtg ggagccagcg
     6121 agtggcttag gaggtccccg agccggagaa tatgcgctgg cttgggcgct ctgtgacctg
     6181 cccccaccct ggcccccaga ctcagaaaag gcctcggctt cgctgcccgg cggctcccca
     6241 gaggacggtt cgctgaaaaa gaagcagcgg cggcagcgca cgcacttcac cagccagcag
     6301 ctacaggagc tagaggcgac cttccagagg aaccgctacc ccgacatgag cacgcgcgag
     6361 gagatcgccg tgtggaccaa cctcaccgag gcccgcgtgc gggtatgctc tccagacccg
     6421 cgactcgcac ccgcgcgggc cctccgcgct cagcctgcga cccggaggcc agcggttcca
     6481 ccccacctgc cccctccctc ggaccccgga ccccggaccc tggctcccgg ccgcagcccc
     6541 acgcggtctc ttgcccgcca acctccagcc tctgctccca ctgctcccta ctactcggac
     6601 cgcgcgcttc ttcggccctg acaccgtctc tccttcccct caccacccct cctcaacccc
     6661 atcctactgt cccgccagac cctggcgccg atgactcggt ccagccgccc gcgccttctg
     6721 ttctccgacc ccggactccg tgcgctcctt ccctcccacc ccgtctctag ccacctcatc
     6781 tcgtttattg accgcaggga gatctgccgg tctccctata gtgagtcgta ttaatttcga
     6841 taagccaggt taacctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     6901 ttgggcgct
//
