LOCUS       sequence-447930-        6286 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting plasmid for inserting a C-terminal AM-Tag in the human 
            HAND1 gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6286)
  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 6286)
  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: pHAND1-AM-Tag-PGK-Neo
FEATURES             Location/Qualifiers
     source          1..6286
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    42..75
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        140..639
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(175..198)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     572..591
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        651..698
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             717..1520
                     /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(780..799)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1390..1409
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1552..1569)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1558..1782
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    1839..1872
                     /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(1888..1904)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1888..1904)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2764..2780)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2764..2780)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2777..2799)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2788..2804
                     /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(2812..2818,2819..2836,2837..2842))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2857..2878
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2995..3012)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3166..3754)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3246..3265)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3925..4716,4717..4785))
                     /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     4548..4567
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4786..4890)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4958..4976
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5014..5036)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5136..5155
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5349..5371
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5363..5380
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5364..5380
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 acaagtgagc ccaattccga tcatattcaa taacccttaa tataacttcg tataatgtat
       61 gctatacgaa gttattaggt ctgaagagga gtttacgtcc agccaagcta gcttggctgc
      121 aggtcgtcga aattctaccg ggtaggggag gcgcttttcc caaggcagtc tggagcatgc
      181 gctttagcag ccccgctggg cacttggcgc tacacaagtg gcctctggcc tcgcacacat
      241 tccacatcca ccggtaggcg ccaaccggct ccgttctttg gtggcccctt cgcgccacct
      301 tctactcctc ccctagtcag gaagttcccc cccgccccgc agctcgcgtc gtgcaggacg
      361 tgacaaatgg aagtagcacg tctcactagt ctcgtgcaga tggacagcac cgctgagcaa
      421 tggaagcggg taggcctttg gggcagcggc caatagcagc tttgctcctt cgctttctgg
      481 gctcagaggc tgggaagggg tgggtccggg ggcgggctca ggggcgggct caggggcggg
      541 gcgggcgccc gaaggtcctc cggaggcccg gcattctgca cgcttcaaaa gcgcacgtct
      601 gccgcgctgt tctcctcttc ctcatctccg ggcctttcga cctgcagcct gttgacaatt
      661 aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac taaaccatgg
      721 gatcggccat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
      781 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
      841 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
      901 aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
      961 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     1021 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     1081 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     1141 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     1201 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc
     1261 ccgacggcga tgatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     1321 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     1381 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     1441 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     1501 ttcttgacga gttcttctga ggggatcaat tctctagagc tcgctgatca gcctcgactg
     1561 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     1621 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     1681 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     1741 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     1801 ccagctgggg ctcgactaga gcttgcggaa cccttaatat aacttcgtat aatgtatgct
     1861 atacgaagtt attaggtccc tcgaggggat ccactagttc tagatcagtg agccgaggcc
     1921 cgcgccgagg acctggccag gccagccact cctgaagccc cgggaggaga ggaaggcagc
     1981 ggcgaacgcc aggctctggg ctccggcgac tggtgctacg catcccgcgg agcttctgct
     2041 gagcgccggc aggtcgtcgg ctgcaaccac acacttggat cgcacgtgca atgtcctttg
     2101 atttttttta atacattaag agaaagagaa atatatatat atccaccccc agcccaaccg
     2161 agggcggccc ttggcggcaa catgcaagaa ggagggactg tcgaacccaa gggctcaaag
     2221 acgcactctt ccaccctttt ggagcgaatt tagaacctca gccctatctc catttcccta
     2281 tctggctctt tctctcttgt ccctccatat gatccgcccc gacgccgtct tctctaatta
     2341 aaatgcaata aggaatcaat tcttttctgc ctgagaaaga gaaccagacg caggaagatg
     2401 aaaggctgcc ctttgttctt cgaatcgtgg tggttttatt ttatttttct ttttgtcgct
     2461 gcacttcctg tttagttcca agggaaacac tttctctctt tctctgtctc tctcttttct
     2521 tccttctttt ccttcctttt tgtttctatc taaataaaag ctttccctgt gttggaaagt
     2581 ttttatgtat ttaaactacc taccatgcct gttgtgctca ggtgtttgtt catcctgcca
     2641 tccccaaccc ttttctacct caagtctgtg tgaccactca caggtacctc gcgaatgcat
     2701 ctagatatcg gatcccgggc ccgtcgactg cagaggcctg catgcaagct tggcgtaatc
     2761 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     2821 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     2881 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     2941 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     3001 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     3061 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     3121 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     3181 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     3241 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     3301 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     3361 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     3421 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     3481 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     3541 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     3601 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     3661 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     3721 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     3781 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     3841 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     3901 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     3961 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     4021 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     4081 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     4141 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     4201 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     4261 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     4321 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     4381 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     4441 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     4501 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     4561 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     4621 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     4681 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     4741 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     4801 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     4861 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     4921 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
     4981 tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     5041 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     5101 gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag
     5161 agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
     5221 gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
     5281 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
     5341 agggttttcc cagtcacgac gttgtaaaac gacggccagt gtgacgaaag ctcatccagg
     5401 gacgagggaa agataaatag gcatcgagtc tctaaacggc tgccccggac tggggatggt
     5461 gcaggccggc ttctttcgag gtgcggcgcc tcgaaagaag ttgttattgg gcggatcccg
     5521 gcccccacgc gtcgccgcct ccttcaagta gcaccgcttg gttcctgctg aaagagcggg
     5581 aaggaggacg ctaggccagc tcgactcccc gctcttttac ctccgtatct gaggctccca
     5641 gctccggctt agggggcccc aagcctgcat ccaagccctg atgtttaaac caggttggga
     5701 ggctggcgga agtgtgtggg gtgcagcgag agcgggagaa ccgtggattt ccacccctcc
     5761 cttccaatat cctgggatct cccttaggtg tcctaaagcc aaatcaacgc gcgtcagttt
     5821 caccagacgc agaacatttt ccttaccaaa cctcccgaag ctgcccagcc catcccgggt
     5881 cccaggaact ccgcgcataa aggcaaattt acgagcctgt ttgcctattt cgcaaacaga
     5941 aatccgtcac ttatttaaag actcccagtt actgctcccg acaccttccc ctcctcctca
     6001 tcttacccca tgccgggcga tcggagcaga aaaattaaaa cctgtctctc ccgcctcttt
     6061 tcttctgtgt gtcctttggc agcagcagca cgaaggtttt cctcctgccc tgggcccagt
     6121 cgagaagagg attaaaggac gcaccggctg gccgcagcaa gtctgggcgc tggagttaaa
     6181 ccagtgccag gatccacaga ggaaaggcaa cgtaatcctt tcacaagctt atgggtgcca
     6241 agatcctcaa cgcaaaggca acgtgatact ctctcaggct tactag
//
