LOCUS       sequence-454038-        6163 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes destablized mCherry under AR8 promoter, and constitutive 
            BFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6163)
  AUTHORS   McNamara HM, Solley SC, Adamson B, Chan MM, Toettcher JE
  TITLE     Recording morphogen signals reveals mechanisms underlying gastruloid
            symmetry breaking.
  JOURNAL   Nat Cell Biol. 2024 Oct 2. doi: 10.1038/s41556-024-01521-9.
  PUBMED    39358450
REFERENCE   2  (bases 1 to 6163)
  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: "Nat Cell 
            Biol. 2024 Oct 2. doi: 10.1038/s41556-024-01521-9."
COMMENT     Sequence Label: pHM129
FEATURES             Location/Qualifiers
     source          1..6163
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   5..39
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    368..416
                     /note="synthetic polyadenylation signal"
     misc_feature    430..521
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     470..489
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     promoter        1038..1069
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     regulatory      1125..1134
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1131..1838
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1276..1294)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1544..1563
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2011..2132
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2022..2041)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2076..2095
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2249..2269)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2254..2583
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2434..2569
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2593..3291
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     repeat_region   3568..3630
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3742..3761)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3861..3883
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3921..3939)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4007..4111
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4112..4180,4181..4972)
                     /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     complement(4330..4349)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5143..5731
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5632..5651
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5885..5902
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6019..6040
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6055..6060,6061..6078,6079..6085)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6093..6109
                     /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)."
     primer_bind     6098..6120
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6117..6133
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6117..6133
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ttaaccctag aaagatagtc tgcgtaaaat tgacgcatgc attcttgaaa tattgctctc
       61 tctttctaaa tagcgcgaat ccgtcgctgt gcatttagga catctcagtc gccgcttgga
      121 gctcccgtga ggcgtgcttg tcaatgcggt aagtgtcact gattttgaac tataacgacc
      181 gcgtgagtca aaatgacgca tgattatctt ttacgtgact tttaagattt aactcatacg
      241 ataattatat tgttatttca tgttctactt acgtgataac ttattatata tatattttct
      301 tgttatagat atcaactaga atgctagcat gggcccagat ctccgaggag gtacttggag
      361 cggccgcaat aaaatatctt tattttcatt acatctgtgt gttggttttt tgtgtgaatc
      421 gatagtacta acatacgctc tccatcaaaa caaaacgaaa caaaacaaac tagcaaaata
      481 ggctgtcccc agtgcaagtg caggtgccag aacatttctc tatcgatagg taccgagctc
      541 ggatccacta gtaacggccg ccagtgtgct ggaattcgcc cttgagctct atctgctgcc
      601 ctaaaatgtg tattccatgg aaatgtctgc ccttctctcc ctagtattgc tgccctaaaa
      661 tgtgtattcc atggaaatgt ctgcccttct ctccctagta tctgctgccc taaaatgtgt
      721 attccatgga aatgtctgcc cttctctccc tagtattgct gccctaaaat gtgtattcca
      781 tggaaatgtc tgcccttctc tccctagtat ctgctgccct aaaatgtgta ttccatggaa
      841 atgtctgccc ttctctccct agtattgctg ccctaaaatg tgtattccat ggaaatgtct
      901 gcccttctct ccctagtatc tgctgcccta aaatgtgtat tccatggaaa tgtctgccct
      961 tctctcccta gtattgctgc cctaaaatgt gtattccatg gaaatgtctg cccttctctc
     1021 cctcgagatc tagactctag agggtatata atggaagctc gaattccagc ttggcattcc
     1081 ggtactgttg gtaaaaagct tggcattccg gtactgttgg taaagccacc atggtgagca
     1141 agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg
     1201 agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg
     1261 agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg
     1321 acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac cccgccgaca
     1381 tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact
     1441 tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca
     1501 tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga
     1561 agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg
     1621 gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct gaggtcaaga
     1681 ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt
     1741 tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa cgcgccgagg
     1801 gccgccactc caccggcggc atggacgagc tgtacaagaa gcttagccat ggcttcccgc
     1861 cggaggtgga ggagcaggat gatggcacgc tgcccatgtc ttgtgcccag gagagcggga
     1921 tggaccgtca ccctgcagcc tgtgcttctg ctaggatcaa tgtgtgataa ttctagagtc
     1981 ggggcggccg gccgcttcga gcagacatga taagatacat tgatgagttt ggacaaacca
     2041 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
     2101 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt
     2161 ttcaggttca gggggaggtg tgggaggcct cgaccaaggc agctgtagat cttagccatc
     2221 tgctttttgc ttgtactgtc gaccctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     2281 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     2341 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     2401 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     2461 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     2521 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     2581 aaaaagctta ccatgagcga gctgattaag gagaacatgc acatgaagct gtacatggag
     2641 ggcaccgtgg acaaccatca cttcaagtgc acatccgagg gcgaaggcaa gccctacgag
     2701 ggcacccaga ccatgagaat caaggtggtc gagggcggcc ctctcccctt cgccttcgac
     2761 atcctggcta ctagcttcct ctacggcagc aagaccttca tcaaccacac ccagggcatc
     2821 cccgacttct tcaagcagtc cttccctgag ggcttcacat gggagagagt caccacatac
     2881 gaagacgggg gcgtgctgac cgctacccag gacaccagcc tccaggacgg ctgcctcatc
     2941 tacaacgtca agatcagagg ggtgaacttc acatccaacg gccctgtgat gcagaagaaa
     3001 acactcggct gggaggcctt caccgagacg ctgtaccccg ctgacggcgg cctggaaggc
     3061 agaaacgaca tggccctgaa gctcgtgggc gggagccatc tgatcgcaaa catcaagacc
     3121 acatatagat ccaagaaacc cgctaagaac ctcaagatgc ctggcgtcta ctatgtggac
     3181 tacagactgg aaagaatcaa ggaggccaac aacgagacct acgtcgagca gcacgaggtg
     3241 gcagtggcca gatactgcga cctccctagc aaactggggc acaagcttaa tgcggccgca
     3301 actcccacct gcaacatgcg tgactaacgc ccgccccacg acccgcagcg cccgaccgaa
     3361 aggagcgcac gaccccatgc atcgatacta gttaaaagtt ttgttacttt atagaagaaa
     3421 ttttgagttt ttgttttttt ttaataaata aataaacata aataaattgt ttgttgaatt
     3481 tattattagt atgtaagtgt aaatataata aaacttaata tctattcaaa ttaataaata
     3541 aacctcgata tacagaccga taaaacacat gcgtcaattt tacgcatgat tatctttaac
     3601 gtacgtcaca atatgattat ctttctaggg ttaatctagt atacgcgtat gcggccgctt
     3661 aattaatcca tggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     3721 ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     3781 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     3841 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     3901 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     3961 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     4021 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     4081 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     4141 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     4201 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     4261 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     4321 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     4381 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     4441 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     4501 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     4561 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     4621 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     4681 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     4741 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     4801 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     4861 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     4921 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     4981 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     5041 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     5101 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     5161 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     5221 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     5281 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     5341 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     5401 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     5461 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     5521 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     5581 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     5641 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     5701 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     5761 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     5821 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     5881 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
     5941 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     6001 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
     6061 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
     6121 aaacagctat gaccatgatt acgccggggc gcgccattct aga
//
