LOCUS       sequence-405820-        5114 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor plasmid to create 5' knockin of NKX2-5 with GFP-T2A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5114)
  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 5114)
  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-GFP-T2A
FEATURES             Location/Qualifiers
     source          1..5114
                     /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"
     regulatory      1225..1234
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1231..1233,1234..1236,1237..1947)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1231..1233,1234..1236,1237..1947)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1276..1297)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1537..1556)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1884..1905
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1957..2010
                     /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"
     primer_bind     complement(2893..2909)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2893..2909)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2906..2928)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2917..2933
                     /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(2941..2947,2948..2965,2966..2971))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2986..3007
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3124..3141)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3295..3883)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3375..3394)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4054..4845,4846..4914))
                     /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     4677..4696
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4915..5019)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5087..5105
                     /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 ccagtgaatt cgagctcggt acctggcagt
      421 gactgatgcc ccctggagtt gtgctgtgca taacactact gtaggaggca gcaactccta
      481 ccccacctgg ccatcactca ccttgccctt actttcgttg actcgcccag aagcacccag
      541 agcctgcggc atgactgacc ctgtaggcca agccaaacca aacccccgaa ttgtccagaa
      601 tctcgccctg ctgctatccc caaagccagc cctgtcctcc aggcttctct tcctattcag
      661 actctccctc atcccaccac cttcagtaat aaagccttcc tcaaatcact ctcctcccca
      721 ccgcttccca ccccatcctt ctctctccca tgctggtttg ggtgctgagg aatatttctt
      781 caaacccaca cccatccagc cctgcccaga ggcctgactc tgcatgcctc tggtaggctt
      841 ttcaggctca cattagggag caaacgcagg ctgcaggagc aaaaggagac ccttccaaat
      901 gcgtcgtggc ccctttaaaa aagctgggca gggctttttt tttttttttt tttttttttt
      961 tttgccgtat gactatatta ggtgacacga aactgctcat cgctcctgtc atcgaggccc
     1021 ctggcccaat ggcaggctga gtccccctcc tctggcctgg tcccgcctct cctgcccctt
     1081 gtgctcagcg ctacctgctg cccggacaca tccagagctg gccgacgggt gcgcgggcgg
     1141 gcggcggcac catgcaggga agctgccagg ggccgtgggc agcgccgctt tctgccgccc
     1201 atctggcgct gtgagactgg cgctgccacc atggtgagca agggcgagga gctgttcacc
     1261 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     1321 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     1381 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
     1441 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     1501 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     1561 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     1621 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     1681 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     1741 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     1801 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     1861 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     1921 actctcggca tggacgagct gtacaagggc agtggagagg gcagaggaag tctgctaaca
     1981 tgcggtgacg tcgaggagaa tcctggccca atgttcccca gccctgctct cacgcccact
     2041 ccgttctcag tcaaagacat cctaaacctg gagcagcagc agcgcagcct ggctgccgcc
     2101 ggagagctct ctgcccgcct ggaggcgacc ctggcgccct cctcctgcat gctggccgcc
     2161 ttcaagccag aggcctacgc tgggcccgag gcggctgcgc cgggcctccc agagctgcgc
     2221 gcagagctgg gccgcgcgcc ttcaccggcc aagtgtgcgt ctgcctttcc cgccgccccc
     2281 gccttctatc cacgtgccta cagcgacccc gacccagcca aggaccctag agccgaaaag
     2341 aaaggtgagg aggaaacaca ggcccccttc tcccctcctg ggtcgctttc gtccccaaga
     2401 aactcagggc caggaggagg agacacgcgc ccttgggccg agggctgggc tgcggcgggg
     2461 ggttcagaat gtaagatgcc tggtgttgtc gccaggctcc cgcgccccgc gtccaatcgg
     2521 aggttcagag gaaatgccgg attgaaagga tcagaagcaa gagaccaaaa aacgtttccc
     2581 cccggcctaa caaagccccg ggcggcttcg gctctgctcc tgggtctggt aggaagttga
     2641 gaaatcggtt tatggtagac agaacagaga gacaagcaga taatctctgt ttttaaatct
     2701 cctttggatt tacgaatctt tttaaagatc tgatgagaac cgctaaacag aaattgaaat
     2761 gttgctcacc agacagcttt tgcgtacaat cggaggaggg tcctggacct tctttctgca
     2821 gcccacccac gacccggggg atcccgggcc cgtcgactgc agaggcctgc atgcaagctt
     2881 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     2941 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     3001 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     3061 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     3121 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     3181 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     3241 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     3301 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     3361 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     3421 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     3481 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     3541 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     3601 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     3661 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     3721 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     3781 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     3841 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     3901 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     3961 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     4021 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     4081 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     4141 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     4201 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     4261 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     4321 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     4381 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     4441 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     4501 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     4561 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     4621 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     4681 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     4741 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     4801 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     4861 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     4921 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     4981 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     5041 acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac
     5101 gaggcccttt cgtc
//
