LOCUS       sequence-171444-        7551 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor vector to knocin EGFP into human Brachyury(T) locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7551)
  AUTHORS   Chu LF, Leng N, Zhang J, Hou Z, Mamott D, Vereide DT, Choi J, 
            Kendziorski C, Stewart R, Thomson JA
  TITLE     Single-cell RNA-seq reveals novel regulators of human embryonic stem
            cell differentiation to definitive endoderm.
  JOURNAL   Genome Biol. 2016 Aug 17;17(1):173. doi: 10.1186/s13059-016-1033-x.
  PUBMED    27534536
REFERENCE   2  (bases 1 to 7551)
  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: "Genome 
            Biol."; date: "2016-08-17"; volume: "17(1):173. doi"; pages: " 
            10.1186/s13059-016-1033-x"
COMMENT     Sequence Label: T-2A-EGFP-PGK-Puro
FEATURES             Location/Qualifiers
     source          1..7551
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             763..819
                     /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             join(820..822,823..825,826..1539)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(820..822,823..825,826..1536)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(865..886)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1126..1145)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1473..1494
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    1570..1603
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1664..2163
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1699..1722)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2096..2115
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2203..2802
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APHNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2203..2222)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     protein_bind    3359..3392
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(3984..4003)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3985..4003)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4010..4027)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4010..4026)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4019..4041)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4167..4595
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4254..4273)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4464..4485
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             4939..5733
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4993..5012)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5603..5622
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(5751..5819,5820..6611)
                     /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(5969..5988)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6782..7370
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7271..7290
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7524..7541
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg
      241 gtaccgagct cggatcccat cttcctgatg attttgtttc ttattaatag atacgacaat
      301 aacttctata catcacagaa tgtgcttgtg agtcctttaa taatttggag aaaatgacct
      361 gtttgaaaga agaaaactgt catattacac agtcactccg aatgggattt ctggtgtgtt
      421 tttccatcct tagctggcct gcagcccctg cccaggccct gctcactggt gtctttctgt
      481 tgcagtcagt accccagcct gtggtctgtg agcaacggcg ccgtcacccc gggctcccag
      541 gcagcagccg tgtccaacgg gctgggggcc cagttcttcc ggggctcccc cgcgcactac
      601 acacccctca cccatccggt ctcggcgccc tcttcctcgg gatccccact gtacgaaggg
      661 gcggccgcgg ccacagacat cgtggacagc cagtacgacg ccgcagccca aggccgcctc
      721 atagcctcat ggacacctgt gtcgccacct tccatggcta gcgccactaa cttctccctg
      781 ttgaaacaag caggggatgt cgaagagaat cccgggccaa tggtgagcaa gggcgaggag
      841 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
      901 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
      961 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     1021 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     1081 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     1141 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     1201 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     1261 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     1321 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     1381 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     1441 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     1501 gccgggatca ctctcggcat ggacgagctg tacaagtaag aattccgatc atattcaata
     1561 acccttaata taacttcgta taatgtatgc tatacgaagt tattaggtct gaagaggagt
     1621 ttacgtccag ccaagcttag gatctcgacc tcgaaattct accgggtagg ggaggcgctt
     1681 ttcccaaggc agtctggagc atgcgcttta gcagccccgc tgggcacttg gcgctacaca
     1741 agtggcctct ggcctcgcac acattccaca tccaccggta ggcgccaacc ggctccgttc
     1801 tttggtggcc ccttcgcgcc accttctact cctcccctag tcaggaagtt cccccccgcc
     1861 ccgcagctcg cgtcgtgcag gacgtgacaa atggaagtag cacgtctcac tagtctcgtg
     1921 cagatggaca gcaccgctga gcaatggaag cgggtaggcc tttggggcag cggccaatag
     1981 cagctttgct ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc cgggggcggg
     2041 ctcaggggcg ggctcagggg cggggcgggc gcccgaaggt cctccggagg cccggcattc
     2101 tgcacgcttc aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc tccgggcctt
     2161 tcgacctgca tccatctaga tctcgagcag ctgaagctta ccatgaccga gtacaagccc
     2221 acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg
     2281 ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc
     2341 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc
     2401 gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg
     2461 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag
     2521 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc
     2581 accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc
     2641 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccac
     2701 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa
     2761 ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gacgcccgcc ccacgacccg
     2821 cagcgcccga ccgaaaggag cgcacgaccc catgcatcga tgatatcaga tccccgggat
     2881 gcagaaattg atgatctatt aaacaataaa gatgtccact aaaatggaag tttttcctgt
     2941 catactttgt taagaagggt gagaacagag tacctacatt ttgaatggaa ggattggagc
     3001 tacgggggtg ggggtggggt gggattagat aaatgcctgc tctttactga aggctcttta
     3061 ctattgcttt atgataatgt ttcatagttg gatatcataa tttaaacaag caaaaccaaa
     3121 ttaagggcca gctcattcct cccactcatg atctatagat ctatagatct ctcgtgggat
     3181 cattgttttt ctcttgattc ccactttgtg gttctaagta ctgtggtttc caaatgtgtc
     3241 agtttcatag cctgaagaac gagatcagca gcctctgttc cacatacact tcattctcag
     3301 tattgttttg ccaagttcta attccatcag aagctggtcg agatccggaa cccttaatat
     3361 aacttcgtat aatgtatgct atacgaagtt attaggtccc tcgaagaggt tcactaggcg
     3421 cgccagcagc aaggcccagg tcccgaaaga tgcagtgact ttttgtcgtg gcagccagtg
     3481 gtgactggat tgacctacta ggtacccagt ggcagtctca ggttaagaag gaaatgcagc
     3541 ctcagtaact tccttttcaa agcagtggag gagcacacgg cacctttccc cagagcccca
     3601 gcatcccttg ctcacacctg cagtagcggt gctgtcccag gtggcttaca gatgaaccca
     3661 actgtggaga tgatgcagtt ggcccaacct cactgacggt gaaaaaatgt ttgccagggt
     3721 ccagaaactt tttttggttt atttctcata cagtgtattg gcaactttgg cacaccagaa
     3781 tttgtaaact ccaccagtcc tactttagtg agataaaaag cacactctta atcttcttcc
     3841 ttgttgcttt caagtagtta gagttgagct gttaaggaca gaataaaatc atagttgagg
     3901 acagggccgg ccaaaagggc gaattctgca gatatccatc acactggcgg ccgctcgagc
     3961 atgcatctag agggcccaat tcgccctata gtgagtcgta ttacaattca ctggccgtcg
     4021 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     4081 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     4141 agttgcgcag cctgaatggc gaatggacgc gccctgtagc ggcgcattaa gcgcggcggg
     4201 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     4261 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     4321 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     4381 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     4441 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     4501 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     4561 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaattcagg gcgcaagggc
     4621 tgctaaagga agcggaacac gtagaaagcc agtccgcaga aacggtgctg accccggatg
     4681 aatgtcagct actgggctat ctggacaagg gaaaacgcaa gcgcaaagag aaagcaggta
     4741 gcttgcagtg ggcttacatg gcgatagcta gactgggcgg ttttatggac agcaagcgaa
     4801 ccggaattgc cagctggggc gccctctggt aaggttggga agccctgcaa agtaaactgg
     4861 atggctttct tgccgccaag gatctgatgg cgcaggggat caagatctga tcaagagaca
     4921 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     4981 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     5041 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     5101 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc
     5161 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     5221 ggcgaagtgc cggggcagga tctcctgtca tcccaccttg ctcctgccga gaaagtatcc
     5281 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     5341 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     5401 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     5461 aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     5521 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     5581 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     5641 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     5701 gccttctatc gccttcttga cgagttcttc tgaattgaaa aaggaagagt atgagtattc
     5761 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     5821 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     5881 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     5941 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     6001 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     6061 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     6121 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     6181 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     6241 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     6301 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     6361 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     6421 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     6481 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     6541 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     6601 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     6661 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     6721 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     6781 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     6841 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     6901 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     6961 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     7021 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     7081 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     7141 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     7201 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     7261 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     7321 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     7381 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     7441 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     7501 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa g
//
