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             1084..1140
                     /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             1147..1857
                     /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(1292..1310)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1560..1579
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    1864..1897
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     promoter        1919..2429
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2348..2366
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2451..3047
                     /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(2451..2470)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2947..2967
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3693..3740
                     /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    3909..3990
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3946..3965)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4000..4019
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    4035..4068
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     rep_origin      complement(5115..5570)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5252..5273)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5464..5483
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5597..5701
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5703..6060
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     primer_bind     complement(5726..5746)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5911..6046
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5973..5992
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             6095..6889
                     /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(6149..6168)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6759..6778
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(7077..7096)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7121..7168
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      7497..8085
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7986..8005
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgtgaattcg agacggtcga
       61 cgctagcggg gttattggta ggtattacta attccacttt aacctgctct taaggcaaac
      121 acacagatga caaacttctc aatggccctg aggactttgc ttaaggaccc aagtgatcgg
      181 caggcagcca agtgctgatc actcgttcca atttggtgtc ttagtctgaa acagtaatct
      241 gccttgattt acccaggctt gctctagagt ccgaatgcct ttgtagatgc cagagggttt
      301 agcactcatt tcagcatcct cgttttatat cctgaaaaac agtcatgggg caaggtcaag
      361 acgcagaact ggtcttcagc ccagctctgg ggtccctggc gtattgcccc atgctgacac
      421 gctaatcccc tgtctactgg acaagcctgt gacagcaatg atggctttct aaagaatatg
      481 cattctgtga atgcggggat tgtggggtgg ggttgggaac acattgccct gagccttggt
      541 gctcagtgct aatcctgtat gccaatgagc acagagctac ttgtcagagt cctctttagg
      601 aggactcggc acactaaaca gtcactgtgc ttatgggcac cttattggtt ttgtttgatg
      661 cagtccatcc ctgtggtcat agtgaaaaaa aaaatagtct gcaaagccct gtgatgcaat
      721 taagtactga ttcctccccc acatctcatc atagctcatt tgcaggcccc tgatgtctgt
      781 tttctctatt tcagccagta tcccagtctc tggtctgtga gcaatggtac catcacccca
      841 ggctcccaga cagctggggt gtccaacggg ctgggagctc agttctttcg aggctcccct
      901 gcacattaca caccactgac acacacggtc tcagctgcca cgtcctcgtc ttctggttct
      961 ccgatgtatg aaggggctgc tacagtcaca gacatttctg acagccagta tgacacggcc
     1021 caaagcctcc tcatagcctc gtggacacct gtgtcacccc catctatgaa gcttggaagc
     1081 ggagctacta acttcagcct gctgaagcag gctggagacg tggaggagaa ccctggacct
     1141 gatatcatgg tgagcaaggg cgaggaggat aacatggcca tcatcaagga gttcatgcgc
     1201 ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc
     1261 gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc
     1321 ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg
     1381 aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg
     1441 gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg
     1501 caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc
     1561 cccgtaatgc agaagaagac catgggctgg gaggcctcct ccgagcggat gtaccccgag
     1621 gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac
     1681 gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac
     1741 aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat cgtggaacag
     1801 tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta caagtaacgt
     1861 acgataactt cgtatagcat acattatacg aagttatatc gatatatcga attcccacgg
     1921 ggttggggtt gcgccttttc caaggcagcc ctgggtttgc gcagggacgc ggctgctctg
     1981 ggcgtggttc cgggaaacgc agcggcgccg accctgggtc tcgcacattc ttcacgtccg
     2041 ttcgcagcgt cacccggatc ttcgccgcta cccttgtggg ccccccggcg acgcttcctg
     2101 ctccgcccct aagtcgggaa ggttccttgc ggttcgcggc gtgccggacg tgacaaacgg
     2161 aagccgcacg tctcactagt accctcgcag acggacagcg ccagggagca atggcagcgc
     2221 gccgaccgcg atgggctgtg gccaatagcg gctgctcagc agggcgcgcc gagagcagcg
     2281 gccgggaagg ggcggtgcgg gaggcggggt gtggggcggt agtgtgggcc ctgttcctgc
     2341 ccgcgcggtg ttccgcattc tgcaagcctc cggagcgcac gtcggcagtc ggctccctcg
     2401 ttgaccgaat caccgacctc tctccccagg gggatccacc ggtcgccacc atgaccgagt
     2461 acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg
     2521 ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg
     2581 agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg
     2641 tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag
     2701 cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg
     2761 ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt
     2821 tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg
     2881 tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg
     2941 cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg
     3001 tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgccgat atctccaaag
     3061 gagaagagct gttcactgga gtggtaccaa tacttgtgga gttggacgga gatgtgaacg
     3121 gacacaaatt ttcagtccgc ggggaggggg aaggggatgc tactattggc aagctgacgc
     3181 tcaaattcat ctgtaccacc ggaaaactcc ctgtaccctg gcccacactg gtgacaactc
     3241 tgacttacgg cgtgcaatgt tttagccgat acccagacca catgaagagg cacgactttt
     3301 tcaaaagcgc aatgcctgaa ggatacgtac aggaaaggac catttctttt aaagacgacg
     3361 ggaagtacaa aacccgggca gtggtgaagt ttgagggcga taccctcgtc aataggatcg
     3421 aattgaaggg aactgacttc aaagaagatg gcaacatcct gggtcacaag cttgagtata
     3481 actttaactc ccacaacgtg tatattacag ccgacaaaca gaagaatgga attaaggcta
     3541 acttcactgt cagacacaat gtcgaagatg gctccgtgca gctcgccgat cactatcaac
     3601 agaatactcc tatcggggac ggcccagtcc tgctgcccga caaccactac ctgagtaccc
     3661 agactgttct gagcaaagat ccgaacgaga agcgcgacca catggtgctg catgagtatg
     3721 tcaacgctgc gggaattacc ctcggcatgg acgagctgta caagtaactc gagttccggt
     3781 gaaacaggcg gccgcgactc tagatcataa tcagccatac cacatttgta gaggttttac
     3841 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg
     3901 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     3961 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     4021 atgtatcttt taaaataact tcgtatagca tacattatac gaagttatta agctctagac
     4081 tcaggtaccg catatgctga gacttgtaac aaccgctgtc aactggatct tctaggctca
     4141 aagtggcagg ctcttgggac aagggaaaaa taaataaata aaagctagat actaacaact
     4201 ccattttcaa ataagagcaa taatacatgt cctataatca tgttctacag cctcttgttt
     4261 gatacctaca gtagtgatat gtgtcctaca ttatgaagcc aaggacagag agacggctgt
     4321 ggtccagttt tttgtgactg gcagttaatc agagtccttt gctaggtagg gtcctatatc
     4381 ttgtgtttct ctacaacata tatgtgactt tgaaatcctg gaattcgtcc accccctgtc
     4441 ctactttagt gagacacaag gtacacctct aatgtcctcc cttgttgcct tagagtagtt
     4501 aactttgagg acagaaaaaa gcatagccag aagattgtaa ctgaaccgtc aactgttctg
     4561 cccttggaac atgcctactt taagcacacg tagctttttg tgttgggaag tcaactgtat
     4621 ggatactttt ctgttgacaa agtagccaaa gacaatctgc agaaagtgtt ttctgcacaa
     4681 taaaggcaat atatagcacc tggtctttgt ggattattct ctacacggct ccttttggga
     4741 aaggctttct tccccagatg tatgttagtc agctgtgaac gttaatgttg tagccaaatc
     4801 agttgcagaa ctgagggttt tcattttgta tacctatgca atctaaggct aaaaaacccc
     4861 aaaaaacaaa gtgaaatgat gtaactttaa gtaacattga atcatagatg aatatgcatg
     4921 gccaaactga aacctgctaa tgtcctaaag tccaggatag acgcctgata caagccactg
     4981 atacataaat gagttgataa aagttggtta ctattaagtc tagttgccat aggtatctgt
     5041 ctgcccatgg ctcttggact cattggctct tttccatcag tggggagatg ctgcagcata
     5101 tgcttaaaag gcgtaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     5161 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     5221 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     5281 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     5341 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     5401 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     5461 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     5521 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac
     5581 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     5641 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     5701 tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     5761 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     5821 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     5881 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     5941 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     6001 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     6061 gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     6121 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     6181 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     6241 caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg
     6301 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     6361 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     6421 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     6481 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     6541 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     6601 actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg
     6661 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     6721 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     6781 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     6841 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     6901 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     6961 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     7021 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag gctaactgaa
     7081 acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat
     7141 aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac
     7201 tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc
     7261 ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca
     7321 ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa acttcatttt
     7381 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     7441 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     7501 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     7561 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     7621 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     7681 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     7741 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     7801 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     7861 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     7921 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     7981 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     8041 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     8101 gcc
//
