LOCUS       sequence-98987-e        7169 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 7169)
  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 7169)
  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..7169
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        384..587
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     538..558
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     632..651
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        632..650
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(731..1351,1352..1735)
                     /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(804..823)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             join(1766..1768,1769..1771,1772..2485)
                     /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(1811..1832)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2532..2549)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2538..2762
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2808..3236
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2895..2914)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3105..3126
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3245..3265)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3250..3579
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3430..3565
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3492..3511
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3646..4440
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3700..3719)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4310..4329
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4614..4735
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4651..4670)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4705..4724
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4784..4800)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4784..4800)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4797..4819)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4808..4824
                     /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(4832..4838,4839..4856,4857..4862))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4877..4898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5015..5032)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5186..5771)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5266..5285)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5942..6733,6734..6802))
                     /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     6565..6584
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6803..6907)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6982..7001)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
      421 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
      481 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
      541 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta
      601 gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag
      661 ctggctagcg tttaaactta agcttggtac cgagctcgga tccgccggcg aaaacctgta
      721 ttttcagctg atgtctagat tagataaaag taaagtgatt aacagcgcat tagagctgct
      781 taatgaggtc ggaatcgaag gtttaacaac ccgtaaactc gcccagaagc taggtgtaga
      841 gcagcctaca ttgtattggc atgtaaaaaa taagcgggct ttgctcgacg ccttagccat
      901 tgagatgtta gataggcacc atactcactt ttgcccttta gaaggggaaa gctggcaaga
      961 ttttttacgt aataacgcta aaagttttag atgtgcttta ctaagtcatc gcgatggagc
     1021 aaaagtacat ttaggtacac ggcctacaga aaaacagtat gaaactctcg aaaatcaatt
     1081 agccttttta tgccaacaag gtttttcact agagaatgca ttatatgcac tcagcgctgt
     1141 ggggcatttt actttaggtt gcgtattgga agatcaagag catcaagtcg ctaaagaaga
     1201 aagggaaaca cctactactg atagtatgcc gccattatta cgacaagcta tcgaattatt
     1261 tgatcaccaa ggtgcagagc cagccttctt attcggcctt gaattgatca tatgcggatt
     1321 agaaaaacaa cttaaatgtg aaagtgggtc cgcgtacagc cgcgcgcgta cgaaaaacaa
     1381 ttacgggtct accatcgagg gcctgctcga tctcccggac gacgacgccc ccgaagaggc
     1441 ggggctggcg gctccgcgcc tgtcctttct ccccgcggga cacacgcgca gactgtcgac
     1501 ggcccccccg accgatgtca gcctggggga cgagctccac ttagacggcg aggacgtggc
     1561 gatggcgcat gccgacgcgc tagacgattt cgatctggac atgttggggg acggggattc
     1621 cccgggtccg ggatttaccc cccacgactc cgccccctac ggcgctctgg atatggccga
     1681 cttcgagttt gagcagatgt ttaccgatgc ccttggaatt gacgagtacg gtggaggtac
     1741 cggcggaggc tccggtggtg gctctatggt gagcaagggc gaggagctgt tcaccggggt
     1801 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgagggg
     1861 cgagggcgag ggcgatgcca ccaacggcaa gctgaccctg aagttcatct gcaccaccgg
     1921 caagctgccc gtgccctggc ccaccctcgt gaccaccctg agccacggcg tgcagtgctt
     1981 cgcccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     2041 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc acctacaaga cccgcgccga
     2101 ggtgaagttc gagggcgaca ccctagtgaa ccgcatcgag ctgaagggcg tcgacttcaa
     2161 ggaggacggc aacatcctgg ggcacaagct ggagtacaac ttcaacagcc acaacatcta
     2221 tatcatggcc gtcaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacgt
     2281 ggaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     2341 ccccgtgctg ctgcccgaca gccactacct gagcacccag tccgtgctga gcaaagaccc
     2401 caacgagaag cgcgatcaca tggtcctgct ggagttccgc accgccgccg ggatcactct
     2461 cggcatggac gagctgtaca agtaatagcg gccgctcgag tctagagggc ccgtttaaac
     2521 ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     2581 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     2641 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     2701 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     2761 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
     2821 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     2881 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     2941 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     3001 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     3061 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     3121 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     3181 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
     3241 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     3301 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     3361 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     3421 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     3481 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
     3541 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
     3601 tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     3661 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     3721 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     3781 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg
     3841 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     3901 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     3961 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     4021 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     4081 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     4141 gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc
     4201 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     4261 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     4321 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     4381 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     4441 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     4501 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     4561 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     4621 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     4681 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     4741 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     4801 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     4861 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     4921 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4981 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     5041 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     5101 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     5161 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     5221 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     5281 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     5341 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     5401 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     5461 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     5521 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     5581 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     5641 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     5701 aacaaaccac cgctggtagc ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     5761 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     5821 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     5881 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     5941 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     6001 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     6061 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     6121 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     6181 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     6241 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     6301 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     6361 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     6421 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     6481 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     6541 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     6601 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     6661 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     6721 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     6781 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     6841 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     6901 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga
     6961 tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct
     7021 gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac
     7081 aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct
     7141 gcttcgcgat gtacgggcca gatatacgc
//
