LOCUS       sequence-454039-        6279 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes rtTA under AR8 promoter, and constitutive BFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6279)
  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 6279)
  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: pHM092
FEATURES             Location/Qualifiers
     source          1..6279
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      17..605
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     506..525
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     759..776
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    893..914
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(929..934,935..952,953..959)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    967..983
                     /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     972..994
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     991..1007
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     991..1007
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   1042..1076
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    1405..1453
                     /note="synthetic polyadenylation signal"
     misc_feature    1467..1558
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     1507..1526
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     promoter        2075..2106
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     CDS             2168..2911
                     /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    3084..3205
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3095..3114)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3149..3168
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3402..3422)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3407..3736
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3587..3722
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3746..4444
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     repeat_region   4721..4783
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4895..4914)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5014..5036
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5074..5092)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5160..5264
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5265..5333,5334..6125)
                     /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(5483..5502)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
       61 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
      121 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
      181 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
      241 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
      301 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
      361 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
      421 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
      481 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
      541 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
      601 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
      661 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
      721 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
      781 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
      841 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
      901 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
      961 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgccg
     1021 gggcgcgcca ttctagatta accctagaaa gatagtctgc gtaaaattga cgcatgcatt
     1081 cttgaaatat tgctctctct ttctaaatag cgcgaatccg tcgctgtgca tttaggacat
     1141 ctcagtcgcc gcttggagct cccgtgaggc gtgcttgtca atgcggtaag tgtcactgat
     1201 tttgaactat aacgaccgcg tgagtcaaaa tgacgcatga ttatctttta cgtgactttt
     1261 aagatttaac tcatacgata attatattgt tatttcatgt tctacttacg tgataactta
     1321 ttatatatat attttcttgt tatagatatc aactagaatg ctagcatggg cccagatctc
     1381 cgaggaggta cttggagcgg ccgcaataaa atatctttat tttcattaca tctgtgtgtt
     1441 ggttttttgt gtgaatcgat agtactaaca tacgctctcc atcaaaacaa aacgaaacaa
     1501 aacaaactag caaaataggc tgtccccagt gcaagtgcag gtgccagaac atttctctat
     1561 cgataggtac cgagctcgga tccactagta acggccgcca gtgtgctgga attcgccctt
     1621 gagctctatc tgctgcccta aaatgtgtat tccatggaaa tgtctgccct tctctcccta
     1681 gtattgctgc cctaaaatgt gtattccatg gaaatgtctg cccttctctc cctagtatct
     1741 gctgccctaa aatgtgtatt ccatggaaat gtctgccctt ctctccctag tattgctgcc
     1801 ctaaaatgtg tattccatgg aaatgtctgc ccttctctcc ctagtatctg ctgccctaaa
     1861 atgtgtattc catggaaatg tctgcccttc tctccctagt attgctgccc taaaatgtgt
     1921 attccatgga aatgtctgcc cttctctccc tagtatctgc tgccctaaaa tgtgtattcc
     1981 atggaaatgt ctgcccttct ctccctagta ttgctgccct aaaatgtgta ttccatggaa
     2041 atgtctgccc ttctctccct cgagatctag actctagagg gtatataatg gaagctcgaa
     2101 ttccagcttg gcattccggt actgttggta aaaagcttgg cattccggta ctgttggtaa
     2161 agccaccatg agccggctgg acaagagcaa agtgatcaac agcgccctgg aactgctgaa
     2221 cggcgtgggc atcgagggcc tgaccacccg gaagctggcc cagaaactgg gcgtggaaca
     2281 gcccaccctg tactggcacg tgaagaacaa gcgggccctg ctggacgccc tgcccatcga
     2341 gatgctggac cggcaccaca cccacagctg ccctctggaa ggcgagagct ggcaggactt
     2401 cctgcggaac aacgccaaga gctacagatg cgccctgctg agccaccggg acggcgccaa
     2461 agtgcacctg ggcaccagac ccaccgagaa gcagtacgag acactggaaa accagctggc
     2521 cttcctgtgc cagcagggct tcagcctgga aaacgccctg tacgccctga gcgccgtggg
     2581 ccacttcacc ctgggctgcg tgctggaaga acaggaacac caggtcgcca aagaggaaag
     2641 agagacaccc accaccgaca gcatgccccc cctgctgaag caggccatcg agctgttcga
     2701 cagacagggc gccgagcccg ccttcctgtt cggcctggaa ctgatcatct gcggcctcga
     2761 gaagcagctg aagtgcgaga gcggcggacc caccgacgcc ctggacgact tcgacctgga
     2821 catgctgccc gccgatgccc tggatgattt tgatctggat atgctccctg ctgacgctct
     2881 cgatgacttc gatctcgata tgctgccagg caagcttagc catggcttcc cgccggaggt
     2941 ggaggagcag gatgatggca cgctgcccat gtcttgtgcc caggagagcg ggatggaccg
     3001 tcaccctgca gcctgtgctt ctgctaggat caatgtgtga taattctaga gtcggggcgg
     3061 ccggccgctt cgagcagaca tgataagata cattgatgag tttggacaaa ccacaactag
     3121 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     3181 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     3241 tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtaaaat
     3301 cgataaggat ccgtcgaccg atgcccttga gagccttcaa cctcgaccaa ggcagctgta
     3361 gatcttagcc atctgctttt tgcttgtact gtcgaccctg tggaatgtgt gtcagttagg
     3421 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     3481 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     3541 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     3601 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     3661 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     3721 cctaggcttt tgcaaaaagc ttaccatgag cgagctgatt aaggagaaca tgcacatgaa
     3781 gctgtacatg gagggcaccg tggacaacca tcacttcaag tgcacatccg agggcgaagg
     3841 caagccctac gagggcaccc agaccatgag aatcaaggtg gtcgagggcg gccctctccc
     3901 cttcgccttc gacatcctgg ctactagctt cctctacggc agcaagacct tcatcaacca
     3961 cacccagggc atccccgact tcttcaagca gtccttccct gagggcttca catgggagag
     4021 agtcaccaca tacgaagacg ggggcgtgct gaccgctacc caggacacca gcctccagga
     4081 cggctgcctc atctacaacg tcaagatcag aggggtgaac ttcacatcca acggccctgt
     4141 gatgcagaag aaaacactcg gctgggaggc cttcaccgag acgctgtacc ccgctgacgg
     4201 cggcctggaa ggcagaaacg acatggccct gaagctcgtg ggcgggagcc atctgatcgc
     4261 aaacatcaag accacatata gatccaagaa acccgctaag aacctcaaga tgcctggcgt
     4321 ctactatgtg gactacagac tggaaagaat caaggaggcc aacaacgaga cctacgtcga
     4381 gcagcacgag gtggcagtgg ccagatactg cgacctccct agcaaactgg ggcacaagct
     4441 taatgcggcc gcaactccca cctgcaacat gcgtgactaa cgcccgcccc acgacccgca
     4501 gcgcccgacc gaaaggagcg cacgacccca tgcatcgata ctagttaaaa gttttgttac
     4561 tttatagaag aaattttgag tttttgtttt tttttaataa ataaataaac ataaataaat
     4621 tgtttgttga atttattatt agtatgtaag tgtaaatata ataaaactta atatctattc
     4681 aaattaataa ataaacctcg atatacagac cgataaaaca catgcgtcaa ttttacgcat
     4741 gattatcttt aacgtacgtc acaatatgat tatctttcta gggttaatct agtatacgcg
     4801 tatgcggccg cttaattaat ccatggcgcc tgatgcggta ttttctcctt acgcatctgt
     4861 gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     4921 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     4981 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     5041 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     5101 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     5161 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     5221 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     5281 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     5341 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     5401 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     5461 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     5521 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     5581 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     5641 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     5701 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     5761 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     5821 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     5881 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     5941 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     6001 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     6061 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     6121 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     6181 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     6241 gtgagttttc gttccactga gcgtcagacc ccgtagaaa
//
