LOCUS       sequence-327655-        7341 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of tTA-EBFP fusion protein (tTA from 
            CL-CTIG, Addgene plasmid 14901).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7341)
  AUTHORS   Daringer NM, Dudek RM, Schwarz KA, Leonard JN
  TITLE     Modular Extracellular Sensor Architecture for Engineering Mammalian 
            Cell-based Devices.
  JOURNAL   ACS Synth Biol. 2014 Mar 11.
  PUBMED    24611683
REFERENCE   2  (bases 1 to 7341)
  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: "ACS Synth 
            Biol. 2014 Mar 11."
COMMENT     Sequence Label: tTA_BFP
FEATURES             Location/Qualifiers
     source          1..7341
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(728..1348,1349..1732)
                     /codon_start=1
                     /product="tetracycline-controlled transactivator, 
                     comprising a fusion of the tetracycline repressor TetR with
                     the C-terminal activation domain of herpes simplex virus 
                     VP16"
                     /label=tTA
                     /note="In the Tet-Off(R) system, tTA binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the absence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTI
                     EGLLDLPDDDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHA
                     DALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
     primer_bind     complement(801..820)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             join(1763..1765,1766..1768,1769..2482)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     complement(1808..1829)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2529..2546)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2535..2759
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2805..3233
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2892..2911)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3102..3123
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3242..3262)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3247..3576
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3427..3562
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3489..3508
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3625..4650
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3625..4650
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    4780..4901
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4817..4836)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4871..4890
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4950..4966)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4950..4966)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4963..4985)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4974..4990
                     /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(4998..5004,5005..5022,5023..5028))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5043..5064
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5181..5198)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5352..5940)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5432..5451)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6111..6902,6903..6971))
                     /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     6734..6753
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6972..7076)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7151..7170)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagcgttt aaacttaagc ttggtaccga gctcggatcc gccggcgaaa acctgtattt
      721 tcagctgatg tctagattag ataaaagtaa agtgattaac agcgcattag agctgcttaa
      781 tgaggtcgga atcgaaggtt taacaacccg taaactcgcc cagaagctag gtgtagagca
      841 gcctacattg tattggcatg taaaaaataa gcgggctttg ctcgacgcct tagccattga
      901 gatgttagat aggcaccata ctcacttttg ccctttagaa ggggaaagct ggcaagattt
      961 tttacgtaat aacgctaaaa gttttagatg tgctttacta agtcatcgcg atggagcaaa
     1021 agtacattta ggtacacggc ctacagaaaa acagtatgaa actctcgaaa atcaattagc
     1081 ctttttatgc caacaaggtt tttcactaga gaatgcatta tatgcactca gcgctgtggg
     1141 gcattttact ttaggttgcg tattggaaga tcaagagcat caagtcgcta aagaagaaag
     1201 ggaaacacct actactgata gtatgccgcc attattacga caagctatcg aattatttga
     1261 tcaccaaggt gcagagccag ccttcttatt cggccttgaa ttgatcatat gcggattaga
     1321 aaaacaactt aaatgtgaaa gtgggtccgc gtacagccgc gcgcgtacga aaaacaatta
     1381 cgggtctacc atcgagggcc tgctcgatct cccggacgac gacgcccccg aagaggcggg
     1441 gctggcggct ccgcgcctgt cctttctccc cgcgggacac acgcgcagac tgtcgacggc
     1501 ccccccgacc gatgtcagcc tgggggacga gctccactta gacggcgagg acgtggcgat
     1561 ggcgcatgcc gacgcgctag acgatttcga tctggacatg ttgggggacg gggattcccc
     1621 gggtccggga tttacccccc acgactccgc cccctacggc gctctggata tggccgactt
     1681 cgagtttgag cagatgttta ccgatgccct tggaattgac gagtacggtg gaggtaccgg
     1741 cggaggctcc ggtggtggct ctatggtgag caagggcgag gagctgttca ccggggtggt
     1801 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgaggggcga
     1861 gggcgagggc gatgccacca acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     1921 gctgcccgtg ccctggccca ccctcgtgac caccctgagc cacggcgtgc agtgcttcgc
     1981 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     2041 cgtccaggag cgcaccatct tcttcaagga cgacggcacc tacaagaccc gcgccgaggt
     2101 gaagttcgag ggcgacaccc tagtgaaccg catcgagctg aagggcgtcg acttcaagga
     2161 ggacggcaac atcctggggc acaagctgga gtacaacttc aacagccaca acatctatat
     2221 catggccgtc aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacgtgga
     2281 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     2341 cgtgctgctg cccgacagcc actacctgag cacccagtcc gtgctgagca aagaccccaa
     2401 cgagaagcgc gatcacatgg tcctgctgga gttccgcacc gccgccggga tcactctcgg
     2461 catggacgag ctgtacaagt aatagcggcc gctcgagtct agagggcccg tttaaacccg
     2521 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     2581 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     2641 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     2701 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     2761 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     2821 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     2881 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     2941 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3001 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     3061 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     3121 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     3181 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg
     3241 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3301 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     3361 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     3421 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     3481 atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag
     3541 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc
     3601 attttcggat ctgatcagca cgtgatgaaa aagcctgaac tcaccgcgac gtctgtcgag
     3661 aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa
     3721 gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc
     3781 tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc
     3841 ccgattccgg aagtgcttga cattggggaa ttcagcgaga gcctgaccta ttgcatctcc
     3901 cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg
     3961 cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg
     4021 ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc
     4081 gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg
     4141 tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg
     4201 cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca
     4261 gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc
     4321 ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg
     4381 catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac
     4441 caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga
     4501 tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga
     4561 agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc
     4621 cccagcactc gtccgagggc aaaggaatag cacgtgctac gagatttcga ttccaccgcc
     4681 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     4741 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     4801 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     4861 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     4921 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     4981 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     5041 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     5101 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5161 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5221 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5281 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5341 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5401 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     5461 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     5521 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     5581 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     5641 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     5701 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     5761 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     5821 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     5881 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     5941 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     6001 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     6061 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     6121 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     6181 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     6241 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     6301 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     6361 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     6421 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     6481 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     6541 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     6601 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6661 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     6721 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     6781 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     6841 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     6901 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     6961 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     7021 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     7081 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt
     7141 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
     7201 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
     7261 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
     7321 tacgggccag atatacgcgt t
//
