LOCUS Exported 5714 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Knock-in of a HaloTag fusion protein into the endogenous OCT4 (Pou5f1) protein. ACCESSION . VERSION . KEYWORDS pKIOct4-Halo-IRES-Bsd SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5714) AUTHORS Strebinger D, Friman ET, Deluz C, Govindan S, Alber AB, Suter DM TITLE Endogenous fluctuations of OCT4 and SOX2 bias pluripotent cell fate decisions JOURNAL bioRxiv 299073 REFERENCE 2 (bases 1 to 5714) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5714 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 371..1261 /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 1274..1294 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="EDLYFQS" misc_feature 1323..1909 /label=IRES2 /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(1490..1507) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 1717..1736 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 1931..2329 /codon_start=1 /gene="Aspergillus terreus BSD" /product="blasticidin S deaminase" /label=BSD /note="confers resistance to blasticidin" /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI KAIVKDSDGQPTAVGIRELLPSGYVWEG" CDS complement(2698..2709) /codon_start=1 /product="WELQut protease recognition and cleavage site" /label=WELQut site /translation="WELQ" rep_origin complement(2726..3181) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2863..2884) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 3075..3094 /label=F1ori-R /note="F1 origin, reverse primer" promoter 3208..3312 /gene="bla" /label=AmpR promoter promoter 3314..3671 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(3337..3357) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 3522..3657 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3584..3603 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3706..4500 /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(3760..3779) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4370..4389 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(4688..4707) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 4732..4779 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 5108..5696 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5597..5616 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgtgaattcg agacggtcga 61 cccatctctg tgcccatcct aggtggttcg agtatggttc tgtaaccggc gccagaaggg 121 caaaagatca agtattgagt attcccaacg agaagagtat gaggctacag ggacaccttt 181 cccagggggg gctgtatcct ttcctctgcc cccaggtccc cactttggca ccccaggcta 241 tggaagcccc cacttcacca cactctactc agtccctttt cctgagggcg aggcctttcc 301 ctctgttccc gtcactgctc tgggctctcc catgcattca aactggcgcg ccgctagccg 361 tctcactagt atggcagaaa tcggtactgg ctttccattc gacccccatt atgtggaagt 421 cctgggcgag cgcatgcact acgtcgatgt tggtccgcgc gatggcaccc ctgtgctgtt 481 cctgcacggt aacccgacct cctcctacgt gtggcgcaac atcatcccgc atgttgcacc 541 gacccatcgc tgcattgctc cagacctgat cggtatgggc aaatccgaca aaccagacct 601 gggttatttc ttcgacgacc acgtccgctt catggatgcc ttcatcgaag ccctgggtct 661 ggaagaggtc gtcctggtca ttcacgactg gggctccgct ctgggtttcc actgggccaa 721 gcgcaatcca gagcgcgtca aaggtattgc atttatggag ttcatccgcc ctatcccgac 781 ctgggacgaa tggccagaat ttgcccgcga gaccttccag gccttccgca ccaccgacgt 841 cggccgcaag ctgatcatcg atcagaacgt ttttatcgag ggtacgctgc cgatgggtgt 901 cgtccgcccg ctgactgaag tcgagatgga ccattaccgc gagccgttcc tgaatcctgt 961 tgaccgcgag ccactgtggc gcttcccaaa cgagctgcca atcgccggtg agccagcgaa 1021 catcgtcgcg ctggtcgaag aatacatgga ctggctgcac cagtcccctg tcccgaagct 1081 gctgttctgg ggcaccccag gcgttctgat cccaccggcc gaagccgctc gcctggccaa 1141 aagcctgcct aactgcaagg ctgtggacat cggcccgggt ctgaatctgc tgcaagaaga 1201 caacccggac ctgatcggca gcgagatcgc gcgctggctg tcgacgctcg agatttccgg 1261 cgagccaacc actgaggatc tgtactttca gagcgataac tctagatgaa gaataaggac 1321 cgcccctctc cctccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg 1381 tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc 1441 cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa 1501 ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga 1561 caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc 1621 ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc 1681 cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac 1741 aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg 1801 tacacatgct ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac 1861 ggggacgtgg ttttcctttg aaaaacacga tgataatatg gccacaacca tgggaagatc 1921 caccgggacc atggccaagc ctttgtctca agaagaatcc accctcattg aaagagcaac 1981 ggctacaatc aacagcatcc ccatctctga agactacagc gtcgccagcg cagctctctc 2041 tagcgacggc cgcatcttca ctggtgtcaa tgtatatcat tttactgggg gaccttgtgc 2101 agaactcgtg gtgctgggca ctgctgctgc tgcggcagct ggcaacctga cttgtatcgt 2161 cgcgatcgga aatgagaaca ggggcatctt gagcccctgc ggacggtgcc gacaggtgct 2221 tctcgatctg catcctggga tcaaagccat agtgaaggac agtgatggac agccgacggc 2281 agttgggatt cgtgaattgc tgccctctgg ttatgtgtgg gagggctaag cggccgcgga 2341 taacttcgta cattatacga agttattggg caccagccct ccctggggat gctgtgagcc 2401 aaggcaaggg aggtagacaa gagaacctgg agctttgggg ttaaattctt ttactgagga 2461 gggattaaaa gcacaacagg ggtggggggt gggatgggga aagaagctca gtgatgctgt 2521 tgatcaggag cctggcctgt ctgtcactca tcattttgtt cttaaataaa gactgggaca 2581 cacagtagat agctgaattt tgttttcctt cagttcctag agagcctgcg gttggagaaa 2641 gccagtaatg gattctcaaa ccccaggtga tcttcaaaac aggcgccatt gaaaccattg 2701 gagttcccat atgcttaaaa ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg 2761 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct 2821 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt 2881 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat 2941 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca 3001 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac 3061 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta 3121 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg 3181 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata 3241 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 3301 aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg 3361 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag 3421 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc 3481 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc 3541 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg 3601 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag 3661 gcttttgcaa agatcgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat 3721 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca 3781 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg 3841 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg 3901 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact 3961 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct 4021 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg 4081 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt 4141 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc 4201 gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc 4261 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga 4321 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc 4381 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt 4441 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga 4501 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt 4561 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg 4621 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cctaggggga 4681 ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa 4741 aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg gttcggtccc 4801 agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt 4861 cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg 4921 tcggggcggc aggccctgcc atagcctcag gttactcata tatactttag attgatttaa 4981 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca 5041 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag 5101 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac 5161 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa 5221 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc 5281 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag 5341 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac 5401 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc 5461 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc 5521 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca 5581 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc 5641 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg 5701 ccagcaacgc ggcc //