LOCUS       sequence-453876-        5932 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes Wnt-responsive rtTA and constitutive BFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5932)
  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 5932)
  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: pHM039
FEATURES             Location/Qualifiers
     source          1..5932
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        156..187
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     CDS             251..994
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     polyA_signal    1167..1288
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1178..1197)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1232..1251
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1485..1505)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1490..1819
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1670..1805
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             1829..2527
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     repeat_region   2804..2866
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2978..2997)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3097..3119
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3157..3175)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3243..3347
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3348..3416,3417..4208)
                     /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(3566..3585)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4379..4967
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4868..4887
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5121..5138
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5255..5276
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5291..5296,5297..5314,5315..5321)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5329..5345
                     /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     5334..5356
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5353..5369
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5353..5369
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   5404..5438
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5767..5815
                     /note="synthetic polyadenylation signal"
     misc_feature    5829..5920
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     5869..5888
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 cgagctctta cgcgagatca aagggggtaa gatcaaaggg ggtaagatca aaggggcgcg
       61 agatcaaagg gggtaagatc aaagggggta agatcaaagg gggtaagatc aaaggggcgc
      121 gcccgcgtgc tagcccgggc tcgagatcta gactctagag ggtatataat ggaagctcga
      181 attccagctt ggcattccgg tactgttggt aaaaagcttg gcattccggt actgttggta
      241 aagccacccc atgagccggc tggacaagag caaagtgatc aacagcgccc tggaactgct
      301 gaacggcgtg ggcatcgagg gcctgaccac ccggaagctg gcccagaaac tgggcgtgga
      361 acagcccacc ctgtactggc acgtgaagaa caagcgggcc ctgctggacg ccctgcccat
      421 cgagatgctg gaccggcacc acacccacag ctgccctctg gaaggcgaga gctggcagga
      481 cttcctgcgg aacaacgcca agagctacag atgcgccctg ctgagccacc gggacggcgc
      541 caaagtgcac ctgggcacca gacccaccga gaagcagtac gagacactgg aaaaccagct
      601 ggccttcctg tgccagcagg gcttcagcct ggaaaacgcc ctgtacgccc tgagcgccgt
      661 gggccacttc accctgggct gcgtgctgga agaacaggaa caccaggtcg ccaaagagga
      721 aagagagaca cccaccaccg acagcatgcc ccccctgctg aagcaggcca tcgagctgtt
      781 cgacagacag ggcgccgagc ccgccttcct gttcggcctg gaactgatca tctgcggcct
      841 cgagaagcag ctgaagtgcg agagcggcgg acccaccgac gccctggacg acttcgacct
      901 ggacatgctg cccgccgatg ccctggatga ttttgatctg gatatgctcc ctgctgacgc
      961 tctcgatgac ttcgatctcg atatgctgcc aggcaagctt agccatggct tcccgccgga
     1021 ggtggaggag caggatgatg gcacgctgcc catgtcttgt gcccaggaga gcgggatgga
     1081 ccgtcaccct gcagcctgtg cttctgctag gatcaatgtg tgataattct agagtcgggg
     1141 cggccggccg cttcgagcag acatgataag atacattgat gagtttggac aaaccacaac
     1201 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     1261 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
     1321 ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtaa
     1381 aatcgataag gatccgtcga ccgatgccct tgagagcctt caacctcgac caaggcagct
     1441 gtagatctta gccatctgct ttttgcttgt actgtcgacc ctgtggaatg tgtgtcagtt
     1501 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     1561 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     1621 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     1681 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     1741 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     1801 aggcctaggc ttttgcaaaa agcttaccat gagcgagctg attaaggaga acatgcacat
     1861 gaagctgtac atggagggca ccgtggacaa ccatcacttc aagtgcacat ccgagggcga
     1921 aggcaagccc tacgagggca cccagaccat gagaatcaag gtggtcgagg gcggccctct
     1981 ccccttcgcc ttcgacatcc tggctactag cttcctctac ggcagcaaga ccttcatcaa
     2041 ccacacccag ggcatccccg acttcttcaa gcagtccttc cctgagggct tcacatggga
     2101 gagagtcacc acatacgaag acgggggcgt gctgaccgct acccaggaca ccagcctcca
     2161 ggacggctgc ctcatctaca acgtcaagat cagaggggtg aacttcacat ccaacggccc
     2221 tgtgatgcag aagaaaacac tcggctggga ggccttcacc gagacgctgt accccgctga
     2281 cggcggcctg gaaggcagaa acgacatggc cctgaagctc gtgggcggga gccatctgat
     2341 cgcaaacatc aagaccacat atagatccaa gaaacccgct aagaacctca agatgcctgg
     2401 cgtctactat gtggactaca gactggaaag aatcaaggag gccaacaacg agacctacgt
     2461 cgagcagcac gaggtggcag tggccagata ctgcgacctc cctagcaaac tggggcacaa
     2521 gcttaatgcg gccgcaactc ccacctgcaa catgcgtgac taacgcccgc cccacgaccc
     2581 gcagcgcccg accgaaagga gcgcacgacc ccatgcatcg atactagtta aaagttttgt
     2641 tactttatag aagaaatttt gagtttttgt ttttttttaa taaataaata aacataaata
     2701 aattgtttgt tgaatttatt attagtatgt aagtgtaaat ataataaaac ttaatatcta
     2761 ttcaaattaa taaataaacc tcgatataca gaccgataaa acacatgcgt caattttacg
     2821 catgattatc tttaacgtac gtcacaatat gattatcttt ctagggttaa tctagtatac
     2881 gcgtatgcgg ccgcttaatt aatccatggc gcctgatgcg gtattttctc cttacgcatc
     2941 tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat
     3001 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     3061 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     3121 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     3181 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     3241 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     3301 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     3361 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     3421 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     3481 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     3541 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     3601 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     3661 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     3721 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     3781 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     3841 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     3901 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     3961 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     4021 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     4081 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     4141 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     4201 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     4261 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     4321 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     4381 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     4441 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     4501 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     4561 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     4621 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     4681 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     4741 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     4801 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     4861 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     4921 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     4981 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     5041 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     5101 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
     5161 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     5221 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
     5281 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
     5341 aacaatttca cacaggaaac agctatgacc atgattacgc cggggcgcgc cattctagat
     5401 taaccctaga aagatagtct gcgtaaaatt gacgcatgca ttcttgaaat attgctctct
     5461 ctttctaaat agcgcgaatc cgtcgctgtg catttaggac atctcagtcg ccgcttggag
     5521 ctcccgtgag gcgtgcttgt caatgcggta agtgtcactg attttgaact ataacgaccg
     5581 cgtgagtcaa aatgacgcat gattatcttt tacgtgactt ttaagattta actcatacga
     5641 taattatatt gttatttcat gttctactta cgtgataact tattatatat atattttctt
     5701 gttatagata tcaactagaa tgctagcatg ggcccagatc tccgaggagg tacttggagc
     5761 ggccgcaata aaatatcttt attttcatta catctgtgtg ttggtttttt gtgtgaatcg
     5821 atagtactaa catacgctct ccatcaaaac aaaacgaaac aaaacaaact agcaaaatag
     5881 gctgtcccca gtgcaagtgc aggtgccaga acatttctct atcgataggt ac
//
