LOCUS       sequence-405818-        6302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor plasmid to create 3' knockin of NKX2-5 with T2A-GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6302)
  AUTHORS   Douglas M, O'Loughlin C, Lynch AT, Prialgauskaite R, Adamson AD, 
            Dibb KM, Kimber SJ, Birket MJ
  TITLE     The generation and validation of two NKX2-5 fluorescent reporter 
            human embryonic stem cell lines: UMANe002-A-1 and UMANe002-A-2.
  JOURNAL   Stem Cell Res. 2023 Dec 3;74:103262. doi: 10.1016/j.scr.2023.103262.
  PUBMED    38100908
REFERENCE   2  (bases 1 to 6302)
  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: "Stem Cell 
            Res."; date: "2023-12-3"; volume: "74:103262. doi"; pages: " 
            10.1016/j.scr.2023.103262"
COMMENT     Sequence Label: pNKX2-5-T2A-GFP-PGK-Puro
FEATURES             Location/Qualifiers
     source          1..6302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1124..1177
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(1178..1180,1181..1894)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1178..1180,1181..1891)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1220..1241)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1481..1500)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1828..1849
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1895..1928)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1929..2428
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1964..1987)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2361..2380
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2444..3043
                     /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(2444..2463)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2940..2960
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(3109..3129)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    complement(3142..3175)
                     /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(4081..4097)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4081..4097)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4094..4116)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4105..4121
                     /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)."
     promoter        complement(join(4129..4135,4136..4153,4154..4159))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4174..4195
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4312..4329)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4483..5071)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4563..4582)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5242..6033,6034..6102))
                     /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     5865..5884
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6103..6207)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6275..6293
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgctta ccattactgt gcggccgcgc
      421 tccgtaggtc aagccgctct taccaagcgt ctctctgcct ctctgttccc cctcagagct
      481 gtgcgcgctg cagaaggcgg tggagctgga gaagacagag gcggacaacg cggagcggcc
      541 ccgggcgcga cggcggagga agccgcgcgt gctcttctcg caggcgcagg tctatgagct
      601 ggagcggcgc ttcaagcagc agcggtacct gtcggccccc gaacgcgacc agctggccag
      661 cgtgctgaaa ctcacgtcca cgcaggtcaa gatctggttc cagaaccggc gctacaagtg
      721 caagcggcag cggcaggacc agactctgga gctggtgggg ctgcccccgc cgccgccgcc
      781 gcctgcccgc aggatcgcgg tgccagtgct ggtgcgcgat ggcaagccat gcctagggga
      841 ctcggcgccc tacgcgcctg cctacggcgt gggcctcaat ccctacggtt ataacgccta
      901 ccccgcctat ccgggttacg gcggcgcggc ctgcagccct ggctacagct gcactgccgc
      961 ttaccccgcc gggccttccc cagcgcagcc ggccactgcc gccgccaaca acaacttcgt
     1021 gaacttcggc gtcggggact tgaatgcggt tcagagcccc gggattccgc agagcaactc
     1081 gggagtgtcc acgctgcatg gtatccgagc ctggggcagt ggagagggca gaggaagtct
     1141 gctaacatgc ggtgacgtcg aggagaatcc tggcccagtg agcaagggcg aggagctgtt
     1201 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     1261 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     1321 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
     1381 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     1441 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     1501 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     1561 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     1621 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     1681 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     1741 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag
     1801 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     1861 gatcactctc ggcatggacg agctgtacaa gtgaataact tcgtatagca tacattatac
     1921 gaagttatgg gtaggggagg cgcttttccc aaggcagtct ggagcatgcg ctttagcagc
     1981 cccgctgggc acttggcgct acacaagtgg cctctggcct cgcacacatt ccacatccac
     2041 cggtaggcgc caaccggctc cgttctttgg tggccccttc gcgccacctt ctactcctcc
     2101 cctagtcagg aagttccccc ccgccccgca gctcgcgtcg tgcaggacgt gacaaatgga
     2161 agtagcacgt ctcactagtc tcgtgcagat ggacagcacc gctgagcaat ggaagcgggt
     2221 aggcctttgg ggcagcggcc aatagcagct ttgctccttc gctttctggg ctcagaggct
     2281 gggaaggggt gggtcccggg gcgggctcag gggcgggctc aggggcgggg cgggcgcccg
     2341 aaggtcctcc ggaggcccgg cattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt
     2401 ctcctcttcc tcatctccgg gcctttcgac ctgcagcgcc accatgaccg agtacaagcc
     2461 cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc
     2521 gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt
     2581 caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt
     2641 cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc
     2701 ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca
     2761 gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc
     2821 caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc
     2881 cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg
     2941 caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga
     3001 aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgagcgatag atcctaatca
     3061 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt
     3121 tacgctatgt ggatacgctg cataacttcg tatagcatac attatacgaa gttatggaag
     3181 ggacccgcgt ggcgcgaccc tgaccgatcc cacctcaaca gctccctgac tctcgggggg
     3241 agaaggggct cccaacatga ccctgagtcc cctggatttt gcattcactc ctgcggagac
     3301 ctaggaactt tttctgtccc acgcgcgttt gttcttgcgc acgggagagt ttgtggcggc
     3361 gattatgcag cgtgcaatga gtgatcctgc agcctggtgt cttagctgtc cccccaggag
     3421 tgccctccga gagtccatgg gcacccccgg ttggaactgg gactgagctc gggcacgcag
     3481 ggcctgagat ctggccgccc attccgcgag ccagggccgg gcgcccgggc ctttgctatc
     3541 tcgccgtcgc ccgcccacgc acccacccgt atttatgttt ttacctattg ctgtaagaaa
     3601 tgacgatccc cttcccatta aagagagtgc gttgaccccg cacgtgtgct tctttcagct
     3661 tgcggcgctt cagaagcagg agagaggtgg ccgcccggga ctggtctcag atctcaggca
     3721 caggcattcc ctgagcaaat tgataacatt gatactaata aaacctaacc cttgctggaa
     3781 ccatactggt tccgtgtcgg gcactttctg agattgtctc atataatcct caataatcca
     3841 aaaaaaaaaa aaatcctaaa gtttagaagc tgaggcccgg agaggtttaa tgacttacct
     3901 gcgagcaaat agccagtact agtcgaactc tggttaaatt caggatgcct cacttcagag
     3961 accgccttcc ctgtgctccc aagctcccct ccttgaatcc gtacctcgcg aatgcatcta
     4021 gatatcggat cccgggcccg tcgactgcag aggcctgcat gcaagcttgg cgtaatcatg
     4081 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     4141 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     4201 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4261 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     4321 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4381 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4441 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4501 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4561 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4621 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4681 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4741 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4801 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     4861 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     4921 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     4981 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5041 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     5101 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5161 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5221 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5281 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5341 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5401 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5461 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5521 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5581 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5641 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     5701 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     5761 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     5821 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     5881 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     5941 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6001 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6061 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6121 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6181 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     6241 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     6301 tc
//
