LOCUS       sequence-447927-        3740 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry vector containing FLAG-HAND1 with a T2A-mTagBFP2 reporter 
            (attL flanked).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3740)
  AUTHORS   Lynch AT, Phillips N, Douglas M, Dorgnach M, Lin IH, Adamson AD, 
            Darieva Z, Whittle J, Hanley NA, Bobola N, Birket MJ
  TITLE     HAND1 level controls the specification of multipotent cardiac and 
            extraembryonic progenitors from human pluripotent stem cells.
  JOURNAL   EMBO J. 2025 Mar 31. doi: 10.1038/s44318-025-00409-0.
  PUBMED    40164946
REFERENCE   2  (bases 1 to 3740)
  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: "EMBO J. 
            2025 Mar 31. doi: 10.1038/s44318-025-00409-0."
COMMENT     Sequence Label: pENTR-FLAG-HAND1-T2A-mTagBFP2
FEATURES             Location/Qualifiers
     source          1..3740
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    16..115
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             124..147
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             205..225
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             826..879
                     /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             880..1587
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDM
                     ALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVA
                     RYCDLPSKLGHKLN"
     protein_bind    complement(1627..1726)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1838..1857)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1849..2658
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPGDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1916..1935)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2748..3336
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3237..3256
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      3504..3547
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3679..3706
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     3714..3737
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
ORIGIN
        1 cttaagctcg ggccccaaat aatgatttta ttttgactga tagtgacctg ttcgttgcaa
       61 caaattgata agcaatgctt ttttataatg ccaactttgt acaaaaaagc aggctccacc
      121 atggattaca aggacgatga cgacaaactc ggatccatgg gaaccaattc agtcgacacc
      181 atgaacctcg tgggcagcta cgcacaccat caccaccatc accacccgca ccctgcgcac
      241 cccatgctcc acgaaccctt cctcttcggt ccggcctcgc gctgtcatca ggaaaggccc
      301 tacttccaga gctggctgct gagcccggct gacgctgccc cggacttccc tgcgggcggg
      361 ccgccgcccg cggccgctgc agccgccacc gcctatggtc ctgacgccag gcctgggcag
      421 agccccgggc ggctggaggc gcttggcggc cgtcttggcc ggcggaaagg ctcaggaccc
      481 aagaaggagc ggagacgcac tgagagcatt aacagcgcat tcgcggagtt gcgcgagtgc
      541 atccccaacg tgccggccga caccaagctc tccaagatca agactctgcg cctagccacc
      601 agctacatcg cctacctgat ggacgtgctg gccaaggatg cacagtctgg cgatcccgag
      661 gccttcaagg ctgaactcaa gaaggcggat ggcggccgtg agagcaagcg gaaaagggag
      721 ctgcagcagc acgaaggttt tcctcctgcc ctgggcccag tcgagaagag gattaaagga
      781 cgcaccggct ggccgcagca agtctgggcg ctggagttaa accaggaggg caggggaagc
      841 ctgctgacat gcggggacgt ggaggaaaat cccggccccg tgtctaaggg cgaagagctg
      901 attaaggaga acatgcacat gaagctgtac atggagggca ccgtggacaa ccatcacttc
      961 aagtgcacat ccgagggcga aggcaagccc tacgagggca cccagaccat gagaatcaag
     1021 gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc tggctactag cttcctctac
     1081 ggcagcaaga ccttcatcaa ccacacccag ggcatccccg acttcttcaa gcagtccttc
     1141 cctgagggct tcacatggga gagagtcacc acatacgaag acgggggcgt gctgaccgct
     1201 acccaggaca ccagcctcca ggacggctgc ctcatctaca acgtcaagat cagaggggtg
     1261 aacttcacat ccaacggccc tgtgatgcag aagaaaacac tcggctggga ggccttcacc
     1321 gagacgctgt accccgctga cggcggcctg gaaggcagaa acgacatggc cctgaagctc
     1381 gtgggcggga gccatctgat cgcaaacgcc aagaccacat atagatccaa gaaacccgct
     1441 aagaacctca agatgcctgg cgtctactat gtggactaca gactggaaag aatcaaggag
     1501 gccaacaacg agacctacgt cgagcagcac gaggtggcag tggccagata ctgcgacctc
     1561 cctagcaaac tggggcacaa gcttaattga gcagtgagaa ttcgcggccg cactcgagat
     1621 atctagaccc agctttcttg tacaaagttg gcattataag aaagcattgc ttatcaattt
     1681 gttgcaacga acaggtcact atcagtcaaa ataaaatcat tatttgccat ccagctgcag
     1741 ctctggcccg tgtctcaaaa tctctgatgt tacattgcac aagataaaaa tatatcatca
     1801 tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt
     1861 caacgggaaa cgtcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat
     1921 aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgctt gtatgggaag
     1981 cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca
     2041 gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat
     2101 tttatccgta ctcctggtga tgcatggtta ctcaccactg cgatccccgg aaaaacagca
     2161 ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg
     2221 ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta
     2281 tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt
     2341 gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca taaacttttg
     2401 ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt
     2461 gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac
     2521 caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg
     2581 ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg
     2641 ctcgatgagt ttttctaatc agaattggtt aattggttgt aacattattc agattgggcc
     2701 ccgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     2761 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     2821 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     2881 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     2941 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     3001 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     3061 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     3121 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     3181 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     3241 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     3301 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     3361 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     3421 tctgtggata accgtattac cgctagcatg gatctcgggg acgtctaact actaagcgag
     3481 agtagggaac tgccaggcat caaataaaac gaaaggccca gtcttccgac tgagcctttc
     3541 gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg ccgggagcgg
     3601 atttgaacgt tgtgaagcaa cggcccggag ggtggcgggc aggacgcccg ccataaactg
     3661 ccaggcatca aactaagcag aaggccatcc tgacggatgg cctttttgcg tttctacaaa
     3721 ctcttcctgt tagttagtta
//
