LOCUS       sequence-447932-        6960 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BFP reporter plasmid targeting the human FOXA2 gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6960)
  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 6960)
  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: pFOXA2-T2A-mTagBFP2-PGK-Neo
FEATURES             Location/Qualifiers
     source          1..6960
                     /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             1226..1279
                     /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             1280..1987
                     /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    2024..2057
                     /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(2157..2180)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2519..2538
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        2598..2645
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2664..3467
                     /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2727..2746)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3337..3356
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3499..3516)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3505..3729
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    3786..3819
                     /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(3835..3851)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3835..3851)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(4739..4755)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4739..4755)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4752..4774)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4763..4779
                     /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(4787..4793,4794..4811,4812..4817))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4832..4853
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4970..4987)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5141..5729)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5221..5240)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5900..6691,6692..6760))
                     /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     6523..6542
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6761..6865)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6933..6951
                     /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 ccagtggctg agcgagatct accagtggat
      421 catggacctc ttccccttct accggcagaa ccagcagcgc tggcagaact ccatccgcca
      481 ctcgctctcc ttcaacgact gtttcctgaa ggtgccccgc tcgcccgaca agcccggcaa
      541 gggctccttc tggaccctgc accctgactc gggcaacatg ttcgagaacg gctgctacct
      601 gcgccgccag aagcgcttca agtgcgagaa gcagctggcg ctgaaggagg ccgcaggcgc
      661 cgccggcagc ggcaagaagg cggccgccgg agcccaggcc tcacaggctc aactcgggga
      721 ggccgccggg ccggcctccg agactccggc gggcaccgag tcgcctcact cgagcgcctc
      781 cccgtgccag gagcacaagc gagggggcct gggagagctg aaggggacgc cggctgcggc
      841 gctgagcccc ccagagccgg cgccctctcc cgggcagcag cagcaggccg cggcccacct
      901 gctgggcccg ccccaccacc cgggcctgcc gcctgaggcc cacctgaagc cggaacacca
      961 ctacgccttc aaccacccgt tctccatcaa caacctcatg tcctcggagc agcagcacca
     1021 ccacagccac caccaccacc agccccacaa aatggacctc aaggcctacg aacaggtgat
     1081 gcactacccc ggctacggtt cccccatgcc tggcagcttg gccatgggcc cggtcacgaa
     1141 caaaacgggc ctggacgcct cgcccctggc cgcagatacc tcctactacc agggggtgta
     1201 ctcccggccc attatgaact cctctgaggg caggggaagc ctgctgacat gcggggacgt
     1261 ggaggaaaat cccggccccg tgtctaaggg cgaagagctg attaaggaga acatgcacat
     1321 gaagctgtac atggagggca ccgtggacaa ccatcacttc aagtgcacat ccgagggcga
     1381 aggcaagccc tacgagggca cccagaccat gagaatcaag gtggtcgagg gcggccctct
     1441 ccccttcgcc ttcgacatcc tggctactag cttcctctac ggcagcaaga ccttcatcaa
     1501 ccacacccag ggcatccccg acttcttcaa gcagtccttc cctgagggct tcacatggga
     1561 gagagtcacc acatacgaag acgggggcgt gctgaccgct acccaggaca ccagcctcca
     1621 ggacggctgc ctcatctaca acgtcaagat cagaggggtg aacttcacat ccaacggccc
     1681 tgtgatgcag aagaaaacac tcggctggga ggccttcacc gagacgctgt accccgctga
     1741 cggcggcctg gaaggcagaa acgacatggc cctgaagctc gtgggcggga gccatctgat
     1801 cgcaaacgcc aagaccacat atagatccaa gaaacccgct aagaacctca agatgcctgg
     1861 cgtctactat gtggactaca gactggaaag aatcaaggag gccaacaacg agacctacgt
     1921 cgagcagcac gaggtggcag tggccagata ctgcgacctc cctagcaaac tggggcacaa
     1981 gcttaattga gcccaattcc gatcatattc aataaccctt aatataactt cgtataatgt
     2041 atgctatacg aagttattag gtctgaagag gagtttacgt ccagccaagc tagcttggct
     2101 gcaggtcgtc gaaattctac cgggtagggg aggcgctttt cccaaggcag tctggagcat
     2161 gcgctttagc agccccgctg ggcacttggc gctacacaag tggcctctgg cctcgcacac
     2221 attccacatc caccggtagg cgccaaccgg ctccgttctt tggtggcccc ttcgcgccac
     2281 cttctactcc tcccctagtc aggaagttcc cccccgcccc gcagctcgcg tcgtgcagga
     2341 cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca gatggacagc accgctgagc
     2401 aatggaagcg ggtaggcctt tggggcagcg gccaatagca gctttgctcc ttcgctttct
     2461 gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg aggcccggca
     2521 ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct cctcttcctc atctccgggc
     2581 ctttcgacct gcagcctgtt gacaattaat catcggcata gtatatcggc atagtataat
     2641 acgacaaggt gaggaactaa accatgggat cggccattga acaagatgga ttgcacgcag
     2701 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     2761 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     2821 agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc
     2881 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     2941 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     3001 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     3061 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     3121 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     3181 tgttcgccag gctcaaggcg cgcatgcccg acggcgatga tctcgtcgtg acccatggcg
     3241 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     3301 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     3361 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     3421 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgaggg gatcaattct
     3481 ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     3541 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     3601 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     3661 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
     3721 gctctatggc ttctgaggcg gaaagaacca gctggggctc gactagagct tgcggaaccc
     3781 ttaatataac ttcgtataat gtatgctata cgaagttatt aggtccctcg aggggatcca
     3841 ctagttctag ataagaagac gacggcttca ggcccggcta actctggcac cccggatcga
     3901 ggacaagtga gagagcaagt gggggtcgag actttgggga gacggtgttg cagagacgca
     3961 agggagaaga aatccataac acccccaccc caacaccccc aagacagcag tcttcttcac
     4021 ccgctgcagc cgttccgtcc caaacagagg gccacacaga taccccacgt tctatataag
     4081 gaggaaaacg ggaaagaata taaagttaaa aaaaagcctc cggtttccac tactgtgtag
     4141 actcctgctt cttcaagcac ctgcagattc tgattttttt gttgttgttg ttctcctcca
     4201 ttgctgttgt tgcagggaag tcttacttaa aaaaaaaaaa aaattttgtg agtgactcgg
     4261 tgtaaaacca tgtagtttta acagaaccag agggttgtac tattgtttaa aaacaggaaa
     4321 aaaaataatg taagggtctg ttgtaaatga ccaagaaaaa gaaaaaaaaa gcattcccaa
     4381 tcttgacacg gtgaaatcca ggtctcgggt ccgattaatt tatggtttct gcgtgcttta
     4441 tttatggctt ataaatgtgt attctggctg caagggccag agttccacaa atctatatta
     4501 aagtgttata cccggtttta tcccttgaat cttttcttcc agatttttct tttctttact
     4561 tggcttacaa aatatacagg cttggaaatt atttcaagaa ggagggaggg ataccctgtc
     4621 tggttgcagg ttgtatttta ttttggccca gggagtgtgt acctcgcgaa tgcatctaga
     4681 tatcggatcc cgggcccgtc gactgcagag gcctgcatgc aagcttggcg taatcatggt
     4741 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     4801 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     4861 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4921 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4981 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     5041 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     5101 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     5161 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     5221 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5281 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5341 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5401 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5461 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     5521 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     5581 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     5641 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     5701 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5761 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5821 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     5881 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5941 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     6001 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     6061 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     6121 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     6181 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     6241 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6301 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6361 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6421 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     6481 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     6541 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     6601 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6661 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6721 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6781 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6841 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag
     6901 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
//
