LOCUS       sequence-178060-        3811 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor vector for ObLiGaRe/ZFN mediated targeting to AAVS1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3811)
  AUTHORS   Pinto R, Hansen L, Hintze J, Almeida R, Larsen S, Coskun M, Davidsen
            J, Mitchelmore C, David L, Troelsen JT, Bennett EP
  TITLE     Precise integration of inducible transcriptional elements (PrIITE) 
            enables absolute control of gene expression.
  JOURNAL   Nucleic Acids Res. 2017 May 4. doi: 10.1093/nar/gkx371.
  PUBMED    28472465
REFERENCE   2  (bases 1 to 3811)
  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: "Nucleic 
            Acids Res. 2017 May 4. doi: 10.1093/nar/gkx371."
COMMENT     Sequence Label: pAAVS1 Insulator ObLiGaRe/EPB71
FEATURES             Location/Qualifiers
     source          1..3811
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(430..448)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(431..448)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1635..1651)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1635..1651)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1648..1670)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1659..1675
                     /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)."
     promoter        complement(join(1683..1689,1690..1707,1708..1713))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1728..1749
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1866..1883)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2037..2625)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2117..2136)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2796..3611)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2796..3611)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3519..3538
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3612..3716)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3784..3802
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtaagct tatcctgtcc ctaggccacc
      421 cactgtgggt ctatagtgtc acctaaatct ctagagccct tcattaggcg cgccaatccc
      481 attgcaaatt ctacaaaagg agtgtttccc aactgctcta tcaagaggaa tgttgcacac
      541 tgtgacctga atgcaaacat cacacgcgcc agcagagagg aagaagagag gcttccctga
      601 ccgggaatcg aacccgggcc gcggcggtga gagcgccgaa tcctaaccac tagaccacca
      661 gggagcacgc gccaaagctc aatgagctat aattatcccc ttggaaaacc tacaaaaaca
      721 gtgtttcaaa actgctctgt gaaaagggac ctttgctagc acgcggcgcc aggcaaaacg
      781 tgggcacgct gcgttggccg ggaatcgaac ccgggtcaac tgcttggaag gcagctatgc
      841 tcaccactat accaccaacg cgcacacgcg ccagcagatt ctacgggaag agtgtttcaa
      901 aactgctcta tcaagagaaa tgttccacct tgtgtgtgga atgcagccat cacacgcgtc
      961 catgaaaggg cttaattaag atatcgttta aacgtcgacc tgcagaggcc ggcggataac
     1021 tagctgatcg cggtcttccc cgacccaggt cacttctggg ttatagctga gactccggac
     1081 agcatgcaac ccattaggcg cgccaatccc attgcaaatt ctacaaaagg agtgtttccc
     1141 aactgctcta tcaagaggaa tgttgcacac tgtgacctga atgcaaacat cacacgcgcc
     1201 agcagagagg aagaagagag gcttccctga ccgggaatcg aacccgggcc gcggcggtga
     1261 gagcgccgaa tcctaaccac tagaccacca gggagcacgc gccaaagctc aatgagctat
     1321 aattatcccc ttggaaaacc tacaaaaaca gtgtttcaaa actgctctgt gaaaagggac
     1381 ctttgctagc acgcggcgcc aggcaaaacg tgggcacgct gcgttggccg ggaatcgaac
     1441 ccgggtcaac tgcttggaag gcagctatgc tcaccactat accaccaacg cgcacacgcg
     1501 ccagcagatt ctacgggaag agtgtttcaa aactgctcta tcaagagaaa tgttccacct
     1561 tgtgtgtgga atgcagccat cacacgcgtc catgaatcct gtccctaggc cacccactgt
     1621 gggtaagctt catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     1681 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     1741 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     1801 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     1861 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     1921 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     1981 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     2041 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     2101 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     2161 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     2221 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     2281 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     2341 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     2401 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     2461 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     2521 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     2581 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     2641 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     2701 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     2761 atctaaagta tatatgagta aacttggtct gacagttaga aaaactcatc gagcatcaaa
     2821 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
     2881 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
     2941 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
     3001 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt
     3061 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
     3121 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga
     3181 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
     3241 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
     3301 ttcccaggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
     3361 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
     3421 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
     3481 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
     3541 tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga
     3601 atatggctca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     3661 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     3721 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     3781 aaaaataggc gtatcacgag gccctttcgt c
//
