LOCUS       sequence-222073-        5714 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Knock-in of a HaloTag fusion protein into the endogenous OCT4 
            (Pou5f1) protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5714)
  AUTHORS   Strebinger D, Deluz C, Friman ET, Govindan S, Alber AB, Suter DM
  TITLE     Endogenous fluctuations of OCT4 and SOX2 bias pluripotent cell fate 
            decisions.
  JOURNAL   Mol Syst Biol. 2019 Sep;15(9):e9002. doi: 10.15252/msb.20199002.
  PUBMED    31556488
REFERENCE   2  (bases 1 to 5714)
  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: "Mol Syst 
            Biol."; date: "2019-09"; volume: "15(9):e9002. doi"; pages: " 
            10.15252/msb.20199002"
COMMENT     Sequence Label: pKIOct4-Halo-IRES-Bsd
FEATURES             Location/Qualifiers
     source          1..5714
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             356..1246
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             1259..1279
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     misc_feature    join(1308..1882,1883..1885,1886..1894)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1475..1492)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1702..1721
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             1916..2314
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(2683..2694)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     rep_origin      complement(2711..3166)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2848..2869)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3060..3079
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3193..3297
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3299..3656
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3299..3490
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3322..3342)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3507..3642
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3691..4485
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3691..4485
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3745..3764)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4355..4374
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4673..4692)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4717..4764
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5093..5681
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5582..5601
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gccttttgct ggccttttgc tcacatgtga attcgagacg gtcgacccat ctctgtgccc
       61 atcctaggtg gttcgagtat ggttctgtaa ccggcgccag aagggcaaaa gatcaagtat
      121 tgagtattcc caacgagaag agtatgaggc tacagggaca cctttcccag ggggggctgt
      181 atcctttcct ctgcccccag gtccccactt tggcacccca ggctatggaa gcccccactt
      241 caccacactc tactcagtcc cttttcctga gggcgaggcc tttccctctg ttcccgtcac
      301 tgctctgggc tctcccatgc attcaaactg gcgcgccgct agccgtctca ctagtatggc
      361 agaaatcggt actggctttc cattcgaccc ccattatgtg gaagtcctgg gcgagcgcat
      421 gcactacgtc gatgttggtc cgcgcgatgg cacccctgtg ctgttcctgc acggtaaccc
      481 gacctcctcc tacgtgtggc gcaacatcat cccgcatgtt gcaccgaccc atcgctgcat
      541 tgctccagac ctgatcggta tgggcaaatc cgacaaacca gacctgggtt atttcttcga
      601 cgaccacgtc cgcttcatgg atgccttcat cgaagccctg ggtctggaag aggtcgtcct
      661 ggtcattcac gactggggct ccgctctggg tttccactgg gccaagcgca atccagagcg
      721 cgtcaaaggt attgcattta tggagttcat ccgccctatc ccgacctggg acgaatggcc
      781 agaatttgcc cgcgagacct tccaggcctt ccgcaccacc gacgtcggcc gcaagctgat
      841 catcgatcag aacgttttta tcgagggtac gctgccgatg ggtgtcgtcc gcccgctgac
      901 tgaagtcgag atggaccatt accgcgagcc gttcctgaat cctgttgacc gcgagccact
      961 gtggcgcttc ccaaacgagc tgccaatcgc cggtgagcca gcgaacatcg tcgcgctggt
     1021 cgaagaatac atggactggc tgcaccagtc ccctgtcccg aagctgctgt tctggggcac
     1081 cccaggcgtt ctgatcccac cggccgaagc cgctcgcctg gccaaaagcc tgcctaactg
     1141 caaggctgtg gacatcggcc cgggtctgaa tctgctgcaa gaagacaacc cggacctgat
     1201 cggcagcgag atcgcgcgct ggctgtcgac gctcgagatt tccggcgagc caaccactga
     1261 ggatctgtac tttcagagcg ataactctag atgaagaata aggaccgccc ctctccctcc
     1321 ccccccccta acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat
     1381 atgttatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct
     1441 gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg
     1501 ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta
     1561 gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag
     1621 ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg
     1681 atagttgtgg aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat
     1741 gcccagaagg taccccattg tatgggatct gatctggggc ctcggtacac atgctttaca
     1801 tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga accacgggga cgtggttttc
     1861 ctttgaaaaa cacgatgata atatggccac aaccatggga agatccaccg ggaccatggc
     1921 caagcctttg tctcaagaag aatccaccct cattgaaaga gcaacggcta caatcaacag
     1981 catccccatc tctgaagact acagcgtcgc cagcgcagct ctctctagcg acggccgcat
     2041 cttcactggt gtcaatgtat atcattttac tgggggacct tgtgcagaac tcgtggtgct
     2101 gggcactgct gctgctgcgg cagctggcaa cctgacttgt atcgtcgcga tcggaaatga
     2161 gaacaggggc atcttgagcc cctgcggacg gtgccgacag gtgcttctcg atctgcatcc
     2221 tgggatcaaa gccatagtga aggacagtga tggacagccg acggcagttg ggattcgtga
     2281 attgctgccc tctggttatg tgtgggaggg ctaagcggcc gcggataact tcgtacatta
     2341 tacgaagtta ttgggcacca gccctccctg gggatgctgt gagccaaggc aagggaggta
     2401 gacaagagaa cctggagctt tggggttaaa ttcttttact gaggagggat taaaagcaca
     2461 acaggggtgg ggggtgggat ggggaaagaa gctcagtgat gctgttgatc aggagcctgg
     2521 cctgtctgtc actcatcatt ttgttcttaa ataaagactg ggacacacag tagatagctg
     2581 aattttgttt tccttcagtt cctagagagc ctgcggttgg agaaagccag taatggattc
     2641 tcaaacccca ggtgatcttc aaaacaggcg ccattgaaac cattggagtt cccatatgct
     2701 taaaaggcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     2761 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     2821 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     2881 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     2941 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     3001 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     3061 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     3121 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt
     3181 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     3241 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtcct
     3301 gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     3361 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     3421 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     3481 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     3541 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     3601 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaagatc
     3661 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     3721 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     3781 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     3841 accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct atcgtggctg
     3901 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     3961 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     4021 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     4081 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     4141 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     4201 ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     4261 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     4321 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     4381 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     4441 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     4501 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     4561 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     4621 agcgcgggga tctcatgctg gagttcttcg cccaccctag ggggaggcta actgaaacac
     4681 ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga cagaataaaa
     4741 cgcacggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg
     4801 tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc ttttccccac
     4861 cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc
     4921 ctgccatagc ctcaggttac tcatatatac tttagattga tttaaaactt catttttaat
     4981 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     5041 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     5101 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     5161 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     5221 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     5281 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     5341 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     5401 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     5461 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     5521 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     5581 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     5641 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     5701 ttttacggtt cctg
//
