LOCUS       sequence-453963-        6336 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes rtTA under IBRE4 promoter, and constitutive BFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6336)
  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 6336)
  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: pHM093
FEATURES             Location/Qualifiers
     source          1..6336
                     /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        1095..1126
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     CDS             1188..1931
                     /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    2104..2225
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2115..2134)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2169..2188
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2422..2442)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2427..2756
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2607..2742
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2766..3464
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     repeat_region   3741..3803
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3915..3934)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4034..4056
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4094..4112)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4180..4284
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4285..4353,4354..5145)
                     /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(4503..4522)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5316..5904
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5805..5824
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6058..6075
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6192..6213
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6228..6233,6234..6251,6252..6258)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6266..6282
                     /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     6271..6293
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6290..6306
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6290..6306
                     /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 ggccgcagac gtggaggagc agagcgccga ggctggcgcc aggcggcctc ttttgtttat
      601 ctgacagcta aatatcaagg caatcaccgc ctcgcggccc tgcctccaac ccccacagct
      661 aagacaaaca gacgcgtggc cgcagacgtg gaggagcaga gcgccgaggc tggcgccagg
      721 cggcctcttt tgtttatctg acagctaaat atcaaggcaa tcaccgcctc gcggccctgc
      781 ctccaacccc cacagctaag acaaacagcc cgggggccgc agacgtggag gagcagagcg
      841 ccgaggctgg cgccaggcgg cctcttttgt ttatctgaca gctaaatatc aaggcaatca
      901 ccgcctcgcg gccctgcctc caacccccac agctaagaca aacagacgcg tggccgcaga
      961 cgtggaggag cagagcgccg aggctggcgc caggcggcct cttttgttta tctgacagct
     1021 aaatatcaag gcaatcaccg cctcgcggcc ctgcctccaa cccccacagc taagacaaac
     1081 agagatctag actctagagg gtatataatg gaagctcgaa ttccagcttg gcattccggt
     1141 actgttggta aaaagcttgg cattccggta ctgttggtaa agccaccatg agccggctgg
     1201 acaagagcaa agtgatcaac agcgccctgg aactgctgaa cggcgtgggc atcgagggcc
     1261 tgaccacccg gaagctggcc cagaaactgg gcgtggaaca gcccaccctg tactggcacg
     1321 tgaagaacaa gcgggccctg ctggacgccc tgcccatcga gatgctggac cggcaccaca
     1381 cccacagctg ccctctggaa ggcgagagct ggcaggactt cctgcggaac aacgccaaga
     1441 gctacagatg cgccctgctg agccaccggg acggcgccaa agtgcacctg ggcaccagac
     1501 ccaccgagaa gcagtacgag acactggaaa accagctggc cttcctgtgc cagcagggct
     1561 tcagcctgga aaacgccctg tacgccctga gcgccgtggg ccacttcacc ctgggctgcg
     1621 tgctggaaga acaggaacac caggtcgcca aagaggaaag agagacaccc accaccgaca
     1681 gcatgccccc cctgctgaag caggccatcg agctgttcga cagacagggc gccgagcccg
     1741 ccttcctgtt cggcctggaa ctgatcatct gcggcctcga gaagcagctg aagtgcgaga
     1801 gcggcggacc caccgacgcc ctggacgact tcgacctgga catgctgccc gccgatgccc
     1861 tggatgattt tgatctggat atgctccctg ctgacgctct cgatgacttc gatctcgata
     1921 tgctgccagg caagcttagc catggcttcc cgccggaggt ggaggagcag gatgatggca
     1981 cgctgcccat gtcttgtgcc caggagagcg ggatggaccg tcaccctgca gcctgtgctt
     2041 ctgctaggat caatgtgtga taattctaga gtcggggcgg ccggccgctt cgagcagaca
     2101 tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct
     2161 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
     2221 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg
     2281 ttttttaaag caagtaaaac ctctacaaat gtggtaaaat cgataaggat ccgtcgaccg
     2341 atgcccttga gagccttcaa cctcgaccaa ggcagctgta gatcttagcc atctgctttt
     2401 tgcttgtact gtcgaccctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     2461 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     2521 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     2581 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     2641 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     2701 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     2761 ttaccatgag cgagctgatt aaggagaaca tgcacatgaa gctgtacatg gagggcaccg
     2821 tggacaacca tcacttcaag tgcacatccg agggcgaagg caagccctac gagggcaccc
     2881 agaccatgag aatcaaggtg gtcgagggcg gccctctccc cttcgccttc gacatcctgg
     2941 ctactagctt cctctacggc agcaagacct tcatcaacca cacccagggc atccccgact
     3001 tcttcaagca gtccttccct gagggcttca catgggagag agtcaccaca tacgaagacg
     3061 ggggcgtgct gaccgctacc caggacacca gcctccagga cggctgcctc atctacaacg
     3121 tcaagatcag aggggtgaac ttcacatcca acggccctgt gatgcagaag aaaacactcg
     3181 gctgggaggc cttcaccgag acgctgtacc ccgctgacgg cggcctggaa ggcagaaacg
     3241 acatggccct gaagctcgtg ggcgggagcc atctgatcgc aaacatcaag accacatata
     3301 gatccaagaa acccgctaag aacctcaaga tgcctggcgt ctactatgtg gactacagac
     3361 tggaaagaat caaggaggcc aacaacgaga cctacgtcga gcagcacgag gtggcagtgg
     3421 ccagatactg cgacctccct agcaaactgg ggcacaagct taatgcggcc gcaactccca
     3481 cctgcaacat gcgtgactaa cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg
     3541 cacgacccca tgcatcgata ctagttaaaa gttttgttac tttatagaag aaattttgag
     3601 tttttgtttt tttttaataa ataaataaac ataaataaat tgtttgttga atttattatt
     3661 agtatgtaag tgtaaatata ataaaactta atatctattc aaattaataa ataaacctcg
     3721 atatacagac cgataaaaca catgcgtcaa ttttacgcat gattatcttt aacgtacgtc
     3781 acaatatgat tatctttcta gggttaatct agtatacgcg tatgcggccg cttaattaat
     3841 ccatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     3901 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     3961 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     4021 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     4081 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     4141 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     4201 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     4261 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     4321 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     4381 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     4441 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     4501 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     4561 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     4621 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     4681 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     4741 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     4801 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     4861 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     4921 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     4981 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     5041 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     5101 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     5161 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     5221 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     5281 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     5341 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     5401 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     5461 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     5521 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     5581 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     5641 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     5701 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     5761 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     5821 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     5881 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     5941 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     6001 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     6061 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
     6121 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     6181 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
     6241 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     6301 tatgaccatg attacgccgg ggcgcgccat tctaga
//
