LOCUS       sequence-81249-e        7484 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector for Human MBD3 conditional knockout. Also creates 
            hypomorphic alleles..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7484)
  AUTHORS   Rais Y, Zviran A, Geula S, Gafni O, Chomsky E, Viukov S, Mansour AA,
            Caspi I, Krupalnik V, Zerbib M, Maza I, Mor N, Baran D, Weinberger 
            L, Jaitin DA, Lara-Astiaso D, Blecher-Gonen R, Shipony Z, Mukamel Z,
            Hagai T, Gilad S, Amann-Zalcenstein D, Tanay A, Amit I, Novershtern 
            N, Hanna JH
  TITLE     Deterministic direct reprogramming of somatic cells to pluripotency.
  JOURNAL   Nature. 2013 Oct 3;502(7469):65-70. doi: 10.1038/nature12587. Epub 
            2013 Sep 18.
  PUBMED    24048479
REFERENCE   2  (bases 1 to 7484)
  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/nature12587"; journalName: "Nature"; date: "2013-10-3- 3"; 
            volume: "502"; issue: "7469"; pages: "65-70"
COMMENT     Sequence Label: pNTK human MBD3 PGK-Neo-pA flox
FEATURES             Location/Qualifiers
     source          1..7484
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        236..298
                     /label=Mini-TK promoter
                     /note="minimal herpes simplex virus (HSV) thymidine kinase 
                     promoter"
     CDS             377..1507
                     /codon_start=1
                     /product="herpes simplex virus thymidine kinase"
                     /label=HSV TK
                     /note="confers sensitivity to nucleoside analogs such as 
                     acyclovir or ganciclovir"
                     /translation="MASYPGHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVRPEQK
                     MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWRVLGASETIANIYTTQ
                     HRLDQGEISAGDAAVVMTSAQITMGMPYTVTDAVLAPHIGGEAGSSHAPPPALTLIFDR
                     HPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQR
                     PGERLDLAMLTAIRRVYGLLANTVRYLQGGGSWREDWGQLSGTAVPPQGAEPQSNAGPR
                     PHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFILDYDQSPAGCRDAL
                     LQLTSGMIQTHVTTPGSIPTICDLARTFAREMGEAN"
     primer_bind     complement(1505..1524)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1549..1597
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     2027..2043
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2584..2617)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3038..3071
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     polyA_signal    3114..3338
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3327..3344
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(3376..4179)
                     /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVNQDDLDE
                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3487..3506)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4097..4116
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4193..4693)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4241..4260)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     4635..4658
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     protein_bind    4714..4747
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    complement(4764..4797)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      5665..5674
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        5718..5822
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5823..5891,5892..6683)
                     /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(6041..6060)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6854..7442
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7343..7362
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cggaccgtag agtcgagcag tgtggttttc aagaggaagc aaaaagcctc tccacccagg
       61 cctggaatgt ttccacccaa tgtcgagcag tgtggttttg caagaggaag caaaaagcct
      121 ctccacccag gcctggaatg tttccaccca atgtcgagca aaccccgccc agcgtcttgt
      181 cattggcgaa ttggaacacg cagatgcagt cggggcggcg cggtcccagg tccacttcgc
      241 atattaaggt gacgcgtgtg gcctcgaaca ccgagcgacc ctgcagcgac ccgcttaaca
      301 gcgtcaacag cgtgccgcac atcttggtgg cgtgaaactc ccgcacctct tcggccagcg
      361 ccttgtagaa gcgcgtatgg cttcgtaccc cggccatcag cacgcgtctg cgttcgacca
      421 ggctgcgcgt tctcgcggcc atagcaaccg acgtacggcg ttgcgccctc gccggcagca
      481 agaagccacg gaagtccgcc cggagcagaa aatgcccacg ctactgcggg tttatataga
      541 cggtccccac gggatgggga aaaccaccac cacgcaactg ctggtggccc tgggttcgcg
      601 cgacgatatc gtctacgtac ccgagccgat gacttactgg cgggtgctgg gggcttccga
      661 gacaatcgcg aacatctaca ccacacaaca ccgccttgac cagggtgaga tatcggccgg
      721 ggacgcggcg gtggtaatga caagcgccca gataacaatg ggcatgcctt ataccgtgac
      781 cgacgccgtt ctggctcctc atatcggggg ggaggctggg agctcacatg ccccgccccc
      841 ggccctcacc ctcatcttcg accgccatcc catcgccgcc ctcctgtgct acccggccgc
      901 gcgatacctt atgggcagca tgacccccca ggccgtgctg gcgttcgtgg ccctcatccc
      961 gccgaccttg cccggcacaa acatcgtgtt gggggccctt ccggaggaca gacacatcga
     1021 ccgcctggcc aaacgccagc gccccggcga gcggcttgac ctggctatgc tgaccgcgat
     1081 tcgccgcgtt tacgggctgc ttgccaatac ggtgcggtat ctgcagggcg gcgggtcgtg
     1141 gcgggaggat tggggacagc tttcggggac ggccgtgccg ccccagggtg ccgagcccca
     1201 gagcaacgcg ggcccacgac cccatatcgg ggacacgtta tttaccctgt ttcgggcccc
     1261 cgagttgctg gcccccaacg gcgacctgta caacgtgttt gcctgggcct tggacgtctt
     1321 ggccaaacgc ctccgtccca tgcacgtctt tatcctggat tacgaccaat cgcccgccgg
     1381 ctgccgggac gccctgctgc aacttacctc cgggatgatc cagacccacg tcaccacccc
     1441 aggctccata ccgacgatct gcgacctggc gcgcacgttt gcacgggaga tgggggaggc
     1501 taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgtcg gcaataaaaa
     1561 gacagaataa aacgcacggg tgttgggtcg tttgttcata aacgcggggt tcggtcccag
     1621 ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct
     1681 tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc
     1741 gggcggcagg cctgccatag ccacgggccc cgtgggttag ggacggggtc ccccatgggg
     1801 aatggtttat ggttcgtggg ggttattatt ttgggcgttg cgtggggtca gtccacgact
     1861 ggactgagca gacagaccca tggtttttgg atggcctggg catggaccgc atgtactggc
     1921 gcgacacgaa caccgggcgt ctgtggctgc caaacacccc cgacccccaa aaaccaccgc
     1981 gcggatttct ggcgccgccg gaccaactaa acctgactac ggaccggtaa aacgacggcc
     2041 agtgttaaca caacccggcc gggaaggctg gggtcgctgt gcgttgagct cacctggcgc
     2101 ggctggtcca ccgccttctg cgggtcgctc ttgaccttgt tgctggggtg gttggtaatc
     2161 ttggtcaccg gctgcttgaa gatggacgcc gtctggcgca cgggcagcgc cgtgttcagg
     2221 tcgggcttgc cctgcgggag gaaggatatg cagtccggcc tgggggcgcc ccggcggcgc
     2281 ggagcctcag ggggtgcggg gcccgggtga ccccctgctt tgccggcgcc cctcgtgtcc
     2341 cccgcgcccg ccaggacccc cacggtccgg ggaggccgct cccgcagggt cggtccggcc
     2401 ggctctggaa cgcccgccgc gggccgcgtc ctcgccatgc gccttgcgct ctgcaccctg
     2461 tggcctgcgc cttccgctct gtgccctccg cccgctgtga cctcctgcgc tcaggacgcc
     2521 gggctgtgtc gcctgcagct cacgtcatgg ccgcgtcccc gcctagaacg ccccgcgaga
     2581 tctataactt cgtatagcat acattatacg aagttataag cttccgacgt cacctgcgtg
     2641 acgccaccac tcaccttgac ctggttggag gagtcgtagc gcacgcgctg gcggctcttg
     2701 ttcatcttgc tcatcagcat cttgcccgtg cggaagtcga aggtgctcag gtccatggag
     2761 ccgcccaggt agcgcgccag ctgcggcttg ctgcggaact tcttcccgct cgggctgcgg
     2821 ggaggcggga cggtcggcgc cccgggcgga ccccagaccc caaacccagg cctcggccgc
     2881 agacccaaac ccagtcccag ccccagcttc aggtcgcgac cccagcccca gaccccaaca
     2941 cggccctgac cccaaaccca ggcaggccct ggccccagcc ccagacccca accctggcgt
     3001 gaccccagac cttcatccca aagctttcta gggtaccgaa gttcctattc tctagaaagt
     3061 ataggaactt cctcgagggg cgcgccccca gctggttctt tccgcctcag aagccataga
     3121 gcccaccgca tccccagcat gcctgctatt gtcttcccaa tcctccccct tgctgtcctg
     3181 ccccacccca ccccccagaa tagaatgaca cctactcaga caatgcgatg caatttcctc
     3241 attttattag gaaaggacag tgggagtggc accttccagg gtcaaggaag gcacggggga
     3301 ggggcaaaca acagatggct ggcaactaga aggcacagtc gaggctgatc agcgagctct
     3361 agagaattga tcccctcaga agaactcgtc aagaaggcga tagaaggcga tgcgctgcga
     3421 atcgggagcg gcgataccgt aaagcacgag gaagcggtca gcccattcgc cgccaagctc
     3481 ttcagcaata tcacgggtag ccaacgctat gtcctgatag cgatccgcca cacccagccg
     3541 gccacagtcg atgaatccag aaaagcggcc attttccacc atgatattcg gcaagcaggc
     3601 atcgccatgg gtcacgacga gatcctcgcc gtcgggcatg cgcgccttga gcctggcgaa
     3661 cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat tgacaagacc
     3721 ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca
     3781 ggtagccgga tcaagcgtat gcagccgccg cattgcatca gccatgatgg atactttctc
     3841 ggcaggagca aggtgagatg acaggagatc ctgccccggc acttcgccca atagcagcca
     3901 gtcccttccc gcttcagtga caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc
     3961 cagccacgat agccgcgctg cctcgtcctg cagttcattc agggcaccgg acaggtcggt
     4021 cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg aacacggcgg catcagagca
     4081 gccgattgtc tgttgtgccc agtcatagcc gaatagcctc tccacccaag cggccggaga
     4141 acctgcgtgc aatccatctt gttcaatggc cgatcccata ttggctgcag gtcgaaaggc
     4201 ccggagatga ggaagaggag aacagcgcgg cagacgtgcg cttttgaagc gtgcagaatg
     4261 ccgggcttcc ggaggacctt cgggcgcccg ccccgcccct gagcccgccc ctgagcccgc
     4321 ccccggaccc accccttccc agcctctgag cccagaaagc gaaggagcca aagctgctat
     4381 tggccgctgc cccaaaggcc tacccgcttc cattgctcag cggtgctgtc catctgcacg
     4441 agactagtga gacgtgctac ttccatttgt cacgtcctgc acgacgcgag ctgcggggcg
     4501 ggggggaact tcctgactag gggaggagta gaaggtggcg cgaaggggcc accaaagaac
     4561 ggagccggtt ggcgcctacc ggtggatgtg gaatgtgtgc gaggccagag gccacttgtg
     4621 tagcgccaag tgcccagcgg ggctgctaaa gcgcatgctc cagactgcct tgggaaaagc
     4681 gcctccccta cccggtaggg cgcgccggga tccgaagttc ctattctcta gaaagtatag
     4741 gaacttcatc gatcctagaa ttcataactt cgtatagcat acattatacg aagttatccg
     4801 cggctagacc cgggggggtc gacctagacc cgggggggtc gagaccccag caccccacaa
     4861 ctggccccta gatctgggcg ccagccagac ccagagcact ggccccgatc ctcacaccct
     4921 ggccctagcc ccagcctcca catcggatcc ttgctccaga cccccaaccc cggtcccctc
     4981 tgaatcctcc ccaccgtagg ccctggcagc gtcaggcaca gcagacgctg agttcgggta
     5041 caaacgctac tacctacagg gctttgggcc ccggctctgg gaggatggct cacatgtcca
     5101 gaacattcca gacatggaat ttaggtcaca ttcttgagaa aagaccccac ggagaacagg
     5161 tatgtgaggg acagccctaa aagcaatctt cttccacctt ggggttacag cagcagggcc
     5221 agactgggaa ggggtctcgt tctaagatct ggggccaggc cctaacagct gcttgggcat
     5281 aacccgactg gagagctcgc aacaaggcag ccaggtaaac acagacctgc tctcagacac
     5341 ggggctctgc tctcagcctg ccatggacaa gcgccggcaa ctcatcctct ccaggacata
     5401 aggacaccca ccaatgggtt gccctgtgtg tcacatgagg tcacactaca ggatgacaag
     5461 cacccaaccc aacacaccgt catcatggtt gggatgtaag atggaacagc catgttggaa
     5521 aagcctgaca gctcttcact gagtttccag atgacccggc cacgccactc ttaggtatct
     5581 gcccgaaaaa tgagcacgca tccacaccat cacttctaca cggatgtccc caggagccag
     5641 aaggtggaag cagccgtcga cgcggccgcc atggtcatag ctgtttgacg tcaggtggca
     5701 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     5761 tgtatccgct cacgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     5821 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     5881 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     5941 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     6001 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     6061 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     6121 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     6181 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     6241 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     6301 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     6361 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     6421 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     6481 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     6541 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     6601 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     6661 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     6721 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     6781 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     6841 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     6901 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     6961 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     7021 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     7081 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     7141 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     7201 tggagcgaac gacctacacc gaactgagat acctacagcg tgagcattga gaaagcgcca
     7261 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     7321 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     7381 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     7441 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgc
//
