LOCUS       sequence-10175-e        6907 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 6907)
  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 6907)
  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-Reverse
FEATURES             Location/Qualifiers
     source          1..6907
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(153..944,945..1013))
                     /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     776..795
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1014..1118)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1186..1204
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1242..1264)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1364..1383
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1464..1482
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1464..1481
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      2336..2345
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2342..2344,2345..2347,2348..3061)
                     /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(2342..2344,2345..2347,2348..3058)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2387..2408)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2648..2667)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2995..3016
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3184..3305
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3221..3240)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3275..3294
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    3350..3383
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(3964..4563)
                     /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(4047..4067)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     4544..4563
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(4603..5099)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4651..4670)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     5041..5064
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     protein_bind    5136..5169
                     /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(6024..6043)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6025..6043)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6130..6147)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6301..6889)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6381..6400)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
       61 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
      121 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
      181 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
      241 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
      301 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
      361 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
      421 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
      481 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
      541 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
      601 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
      661 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
      721 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
      781 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
      841 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
      901 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
      961 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     1021 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     1081 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     1141 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     1201 aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     1261 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     1321 gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt
     1381 gtactgagag tgcaccatat ggacatattg tcgttagaac gcggctacaa ttaatacata
     1441 accttatgta tcatacacat acgatttagg tgacactata gaactcgagc aggtatgatt
     1501 ggctgaggac catgtatgag tataggggga tactaagcat tcaggtgaga aggattttaa
     1561 gtttttgcag atgtagtata gggtatgtag cgtgctatag tgtagatttt gttgttgttg
     1621 ttttttgaga cagcatcttg ctctgtcacc caggctggag tgcagtagca ccatctcggc
     1681 tcgctgcaac ctccgcctcc caggttcaag cgattctcct gcctcagcct cccgagtagc
     1741 tgagattaca ggcacgtgcc accccgccca gcaatttttg tatttttagt agggacggga
     1801 ttttaccatg ttggccaggc tggtctcgaa ctcctgacct caggtgatcc acccgccttg
     1861 gcctcccaaa gtgctgggat tacaggcgtg agccactgcg cccggcctat agtctacatt
     1921 tttattaatt caataaatat tgaaccccaa ctgtatacct ggcacagttc tagttcccag
     1981 cagtaaacga aacgaagtct agtttatata tctatgcaga gaacctctca gcatgcacct
     2041 ctgcgtcctt aggatgctat ttcccgattc tatcctaagg agccccacgc agccccagct
     2101 ttacggctgg ggttgagaaa ggcgccaggg aatttacgag gaaacgctgc tgggacctag
     2161 ctaagccgga gattttaccc ccattaccct ggtctgtgtc ttctctcgcc tccagagccc
     2221 tccctccatg gagttcggcc tgctcagcga ggcagaggcc cggagccctg ccctgtcgct
     2281 gtcagacgct ggcactccgc acccccagct cccagagcac ggggatccac cggtcgccac
     2341 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     2401 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     2461 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     2521 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     2581 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     2641 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     2701 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     2761 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     2821 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     2881 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     2941 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     3001 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta
     3061 aagcggccgc gactctagat cataatcagc cataccacat ttgtagaggt tttacttgct
     3121 ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     3181 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     3241 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     3301 tcttaagggt accgagctcg aattccgatc atattcaata acccttaata taacttcgta
     3361 taatgtatgc tatacgaagt tattaggtct gaagaggagt ttacgtccag ccaagcttag
     3421 gatctcgacc agcttctgat ggaattagaa cttggcaaaa caatactgag aatgaagtgt
     3481 atgtggaaca gaggctgctg atctcgttct tcaggctatg aaactgacac atttggaaac
     3541 cacagtactt agaaccacaa agtgggaatc aagagaaaaa caatgatccc acgagagatc
     3601 tatagatcta tagatcatga gtgggaggaa tgagctggcc cttaatttgg ttttgcttgt
     3661 ttaaattatg atatccaact atgaaacatt atcataaagc aatagtaaag agccttcagt
     3721 aaagagcagg catttatcta atcccacccc acccccaccc ccgtagctcc aatccttcca
     3781 ttcaaaatgt aggtactctg ttctcaccct tcttaacaaa gtatgacagg aaaaacttcc
     3841 attttagtgg acatctttat tgtttaatag atcatcaatt tctgcatccc ggggatctga
     3901 tatcatcgat gcatggggtc gtgcgctcct ttcggtcggg cgctgcgggt cgtggggcgg
     3961 gcgtcaggca ccgggcttgc gggtcatgca ccaggtgcgc ggtccttcgg gcacctcgac
     4021 gtcggcggtg acggtgaagc cgagccgctc gtagaagggg aggttgcggg gcgcggaggt
     4081 ctccaggaag gcgggcaccc cggcgcgctc ggccgcctcc actccgggga gcacgacggc
     4141 gctgcccaga cccttgccct ggtggtcggg cgagacgccg acggtggcca ggaaccacgc
     4201 gggctccttg ggccggtgcg gcgccaggag gccttccatc tgttgctgcg cggccagccg
     4261 ggaaccgctc aactcggcca tgcgcgggcc gatctcggcg aacaccgccc ccgcttcgac
     4321 gctctccggc gtggtccaga ccgccaccgc ggcgccgtcg tccgcgaccc acaccttgcc
     4381 gatgtcgagc ccgacgcgcg tgaggaagag ttcttgcagc tcggtgaccc gctcgatgtg
     4441 gcggtccgga tcgacggtgt ggcgcgtggc ggggtagtcg gcgaacgcgg cggcgagggt
     4501 gcgtacggcc ctggggacgt cgtcgcgggt ggcgaggcgc accgtgggct tgtactcggt
     4561 catggtaagc ttcagctgct cgagatctag atggatgcag gtcgaaaggc ccggagatga
     4621 ggaagaggag aacagcgcgg cagacgtgcg cttttgaagc gtgcagaatg ccgggcctcc
     4681 ggaggacctt cgggcgcccg ccccgcccct gagcccgccc ctgagcccgc ccccggaccc
     4741 acccttccca gctgctgagc ccagaaagcg aaggagcaaa gctgctattg gccgctgccc
     4801 caaaggccta cccgcttcca ttgctcagcg gtgctgtcca tctgcacgag actagtgaga
     4861 cgtgctactt ccatttgtca cgtcctgcac gacgcgagct gcggggcggg ggggaacttc
     4921 ctgactaggg gaggagtaga aggtggcgcg aaggggccac caaagaacgg agccggttgg
     4981 cgctaccggt ggatgtggaa tgtgtgcgag gccagaggcc acttgtgtag cgccaagtgc
     5041 cagcggggct gctaaagcgc atgctccaga ctgccttggg aaaagcgcct cccctacccg
     5101 gtagaatttc gaggtcgaga tccggaaccc ttaatataac ttcgtataat gtatgctata
     5161 cgaagttatt aggtccctcg aagaggttca ctagtactgg ccattgcggc catcctgcaa
     5221 gggccaggag cacagcggta agcgcgcccc cttccggggg tgcaggacat aacagcttca
     5281 tccccagtgc gggtgggacc ccggctgggc agccccggtg ggagccagcg agtggcttag
     5341 gaggtccccg agccggagaa tatgcgctgg cttgggcgct ctgtgacctg cccccaccct
     5401 ggcccccaga ctcagaaaag gcctcggctt cgctgcccgg cggctcccca gaggacggtt
     5461 cgctgaaaaa gaagcagcgg cggcagcgca cgcacttcac cagccagcag ctacaggagc
     5521 tagaggcgac cttccagagg aaccgctacc ccgacatgag cacgcgcgag gagatcgccg
     5581 tgtggaccaa cctcaccgag gcccgcgtgc gggtatgctc tccagacccg cgactcgcac
     5641 ccgcgcgggc cctccgcgct cagcctgcga cccggaggcc agcggttcca ccccacctgc
     5701 cccctccctc ggaccccgga ccccggaccc tggctcccgg ccgcagcccc acgcggtctc
     5761 ttgcccgcca acctccagcc tctgctccca ctgctcccta ctactcggac cgcgcgcttc
     5821 ttcggccctg acaccgtctc tccttcccct caccacccct cctcaacccc atcctactgt
     5881 cccgccagac cctggcgccg atgactcggt ccagccgccc gcgccttctg ttctccgacc
     5941 ccggactccg tgcgctcctt ccctcccacc ccgtctctag ccacctcatc tcgtttattg
     6001 accgcaggga gatctgccgg tctccctata gtgagtcgta ttaatttcga taagccaggt
     6061 taacctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     6121 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     6181 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     6241 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     6301 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     6361 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     6421 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     6481 cgtggcgctt tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     6541 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     6601 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     6661 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     6721 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
     6781 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     6841 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     6901 gatcttt
//
