LOCUS Exported 8103 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Knock-in of P2A-mCherry at the mouse Brachyury (T) gene. ACCESSION . VERSION . KEYWORDS pKIBra-P2A-mCherry-Puro-sfGFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8103) AUTHORS Deluz C, Friman ET, Strebinger D, Benke A, Raccaud M, Callegari A, Leleu M, Manley S, Suter DM TITLE A role for mitotic bookmarking of SOX2 in pluripotency and differentiation. JOURNAL Genes Dev. 2016 Nov 15;30(22):2538-2550. doi: 10.1101/gad.289256.116. Epub 2016 Dec 5. PUBMED 27920086 REFERENCE 2 (bases 1 to 8103) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8103 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 1014..1070 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 1077..1787 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA EGRHSTGGMDELYK" primer_bind complement(1222..1240) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 1490..1509 /label=mCherry-F /note="mCherry, forward primer" protein_bind 1794..1827 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 1849..2359 /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" primer_bind 2278..2296 /label=hPGK-F /note="Human PGK promoter, forward primer" CDS 2381..2977 /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(2381..2400) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 2877..2897 /label=Puro-F /note="Puromycin resistance gene, forward primer" CDS 3623..3670 /codon_start=1 /product="11th beta-strand of superfolder GFP" /label=GFP11 /note="associates with GFP(1-10), consisting of the first 10 beta-strands, to reconstitute fluorescent GFP (Cabantous et al., 2005; Kamiyama et al., 2016)" /translation="RDHMVLHEYVNAAGIT" polyA_signal 3839..3920 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3876..3895) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3930..3949 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" protein_bind 3965..3998 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." rep_origin complement(5045..5500) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5182..5203) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 5394..5413 /label=F1ori-R /note="F1 origin, reverse primer" promoter 5527..5631 /gene="bla" /label=AmpR promoter promoter 5633..5990 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(5656..5676) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 5841..5976 /label=SV40 ori /note="SV40 origin of replication" primer_bind 5903..5922 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 6025..6819 /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(6079..6098) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 6689..6708 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(7007..7026) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" rep_origin 7427..8015 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7916..7935 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 gttattggta ggtattacta attccacttt aacctgctct taaggcaaac acacagatga 61 caaacttctc aatggccctg aggactttgc ttaaggaccc aagtgatcgg caggcagcca 121 agtgctgatc actcgttcca atttggtgtc ttagtctgaa acagtaatct gccttgattt 181 acccaggctt gctctagagt ccgaatgcct ttgtagatgc cagagggttt agcactcatt 241 tcagcatcct cgttttatat cctgaaaaac agtcatgggg caaggtcaag acgcagaact 301 ggtcttcagc ccagctctgg ggtccctggc gtattgcccc atgctgacac gctaatcccc 361 tgtctactgg acaagcctgt gacagcaatg atggctttct aaagaatatg cattctgtga 421 atgcggggat tgtggggtgg ggttgggaac acattgccct gagccttggt gctcagtgct 481 aatcctgtat gccaatgagc acagagctac ttgtcagagt cctctttagg aggactcggc 541 acactaaaca gtcactgtgc ttatgggcac cttattggtt ttgtttgatg cagtccatcc 601 ctgtggtcat agtgaaaaaa aaaatagtct gcaaagccct gtgatgcaat taagtactga 661 ttcctccccc acatctcatc atagctcatt tgcaggcccc tgatgtctgt tttctctatt 721 tcagccagta tcccagtctc tggtctgtga gcaatggtac catcacccca ggctcccaga 781 cagctggggt gtccaacggg ctgggagctc agttctttcg aggctcccct gcacattaca 841 caccactgac acacacggtc tcagctgcca cgtcctcgtc ttctggttct ccgatgtatg 901 aaggggctgc tacagtcaca gacatttctg acagccagta tgacacggcc caaagcctcc 961 tcatagcctc gtggacacct gtgtcacccc catctatgaa gcttggaagc ggagctacta 1021 acttcagcct gctgaagcag gctggagacg tggaggagaa ccctggacct gatatcatgg 1081 tgagcaaggg cgaggaggat aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc 1141 acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc 1201 cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc ctgcccttcg 1261 cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg aagcaccccg 1321 ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga 1381 tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg caggacggcg 1441 agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc 1501 agaagaagac catgggctgg gaggcctcct ccgagcggat gtaccccgag gacggcgccc 1561 tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac gacgctgagg 1621 tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca 1681 tcaagttgga catcacctcc cacaacgagg actacaccat cgtggaacag tacgaacgcg 1741 ccgagggccg ccactccacc ggcggcatgg acgagctgta caagtaacgt acgataactt 1801 cgtatagcat acattatacg aagttatatc gatatatcga attcccacgg ggttggggtt 1861 gcgccttttc caaggcagcc ctgggtttgc gcagggacgc ggctgctctg ggcgtggttc 1921 cgggaaacgc agcggcgccg accctgggtc tcgcacattc ttcacgtccg ttcgcagcgt 1981 cacccggatc ttcgccgcta cccttgtggg ccccccggcg acgcttcctg ctccgcccct 2041 aagtcgggaa ggttccttgc ggttcgcggc gtgccggacg tgacaaacgg aagccgcacg 2101 tctcactagt accctcgcag acggacagcg ccagggagca atggcagcgc gccgaccgcg 2161 atgggctgtg gccaatagcg gctgctcagc agggcgcgcc gagagcagcg gccgggaagg 2221 ggcggtgcgg gaggcggggt gtggggcggt agtgtgggcc ctgttcctgc ccgcgcggtg 2281 ttccgcattc tgcaagcctc cggagcgcac gtcggcagtc ggctccctcg ttgaccgaat 2341 caccgacctc tctccccagg gggatccacc ggtcgccacc atgaccgagt acaagcccac 2401 ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt 2461 cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac 2521 cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc 2581 ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt 2641 gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca 2701 acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac 2761 cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg 2821 agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa 2881 cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg 2941 accgcgcacc tggtgcatga cccgcaagcc cggtgccgat atctccaaag gagaagagct 3001 gttcactgga gtggtaccaa tacttgtgga gttggacgga gatgtgaacg gacacaaatt 3061 ttcagtccgc ggggaggggg aaggggatgc tactattggc aagctgacgc tcaaattcat 3121 ctgtaccacc ggaaaactcc ctgtaccctg gcccacactg gtgacaactc tgacttacgg 3181 cgtgcaatgt tttagccgat acccagacca catgaagagg cacgactttt tcaaaagcgc 3241 aatgcctgaa ggatacgtac aggaaaggac catttctttt aaagacgacg ggaagtacaa 3301 aacccgggca gtggtgaagt ttgagggcga taccctcgtc aataggatcg aattgaaggg 3361 aactgacttc aaagaagatg gcaacatcct gggtcacaag cttgagtata actttaactc 3421 ccacaacgtg tatattacag ccgacaaaca gaagaatgga attaaggcta acttcactgt 3481 cagacacaat gtcgaagatg gctccgtgca gctcgccgat cactatcaac agaatactcc 3541 tatcggggac ggcccagtcc tgctgcccga caaccactac ctgagtaccc agactgttct 3601 gagcaaagat ccgaacgaga agcgcgacca catggtgctg catgagtatg tcaacgctgc 3661 gggaattacc ctcggcatgg acgagctgta caagtaactc gagttccggt gaaacaggcg 3721 gccgcgactc tagatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa 3781 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa 3841 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa 3901 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatcttt 3961 taaaataact tcgtatagca tacattatac gaagttatta agctctagac tcaggtaccg 4021 catatgctga gacttgtaac aaccgctgtc aactggatct tctaggctca aagtggcagg 4081 ctcttgggac aagggaaaaa taaataaata aaagctagat actaacaact ccattttcaa 4141 ataagagcaa taatacatgt cctataatca tgttctacag cctcttgttt gatacctaca 4201 gtagtgatat gtgtcctaca ttatgaagcc aaggacagag agacggctgt ggtccagttt 4261 tttgtgactg gcagttaatc agagtccttt gctaggtagg gtcctatatc ttgtgtttct 4321 ctacaacata tatgtgactt tgaaatcctg gaattcgtcc accccctgtc ctactttagt 4381 gagacacaag gtacacctct aatgtcctcc cttgttgcct tagagtagtt aactttgagg 4441 acagaaaaaa gcatagccag aagattgtaa ctgaaccgtc aactgttctg cccttggaac 4501 atgcctactt taagcacacg tagctttttg tgttgggaag tcaactgtat ggatactttt 4561 ctgttgacaa agtagccaaa gacaatctgc agaaagtgtt ttctgcacaa taaaggcaat 4621 atatagcacc tggtctttgt ggattattct ctacacggct ccttttggga aaggctttct 4681 tccccagatg tatgttagtc agctgtgaac gttaatgttg tagccaaatc agttgcagaa 4741 ctgagggttt tcattttgta tacctatgca atctaaggct aaaaaacccc aaaaaacaaa 4801 gtgaaatgat gtaactttaa gtaacattga atcatagatg aatatgcatg gccaaactga 4861 aacctgctaa tgtcctaaag tccaggatag acgcctgata caagccactg atacataaat 4921 gagttgataa aagttggtta ctattaagtc tagttgccat aggtatctgt ctgcccatgg 4981 ctcttggact cattggctct tttccatcag tggggagatg ctgcagcata tgcttaaaag 5041 gcgtaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc 5101 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc 5161 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac 5221 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca 5281 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg 5341 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag 5401 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc 5461 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac ttttcgggga 5521 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc 5581 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tcctgaggcg 5641 gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag 5701 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc 5761 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag 5821 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc 5881 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc 5941 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa 6001 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 6061 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 6121 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 6181 ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg 6241 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 6301 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 6361 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 6421 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 6481 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 6541 aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc 6601 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 6661 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 6721 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 6781 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa 6841 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct 6901 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg 6961 gggatctcat gctggagttc ttcgcccacc ctagggggag gctaactgaa acacggaagg 7021 agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcgcg 7081 gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata 7141 ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc 7201 ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca 7261 tagcctcagg ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa 7321 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt 7381 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt 7441 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt 7501 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga 7561 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag 7621 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata 7681 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg 7741 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga 7801 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca 7861 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa 7921 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt 7981 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac 8041 ggttcctggc cttttgctgg ccttttgctc acatgtgaat tcgagacggt cgacgctagc 8101 ggg //