LOCUS       sequence-311963-        5581 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Soluble tTA transcription factor flanked by N and C terminal TEV 
            protease G cleavage sequences and 3 nuclear export sequences.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5581)
  AUTHORS   Dolberg TB, Meger AT, Boucher JD, Corcoran WK, Schauer EE, Prybutok 
            AN, Raman S, Leonard JN
  TITLE     Computation-guided optimization of split protein systems.
  JOURNAL   Nat Chem Biol. 2021 May;17(5):531-539. doi: 
            10.1038/s41589-020-00729-8. Epub 2021 Feb 1.
  PUBMED    33526893
REFERENCE   2  (bases 1 to 5581)
  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"; doi: 
            "10.1038/s41589-020-00729-8"; journalName: "Nat Chem Biol"; date: 
            "2021-05"; volume: "17"; issue: "5"; pages: "531-539"
COMMENT     Sequence Label: 3NES-CS(G)-tTA-CS(G)-3NES
FEATURES             Location/Qualifiers
     source          1..5581
                     /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             781..801
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(802..1422,1423..1803)
                     /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
                     DALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYG"
     primer_bind     complement(875..894)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             1804..1824
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1977..1994)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1983..2207
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2253..2681
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2340..2359)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2550..2571
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2704..2900
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2751..2886
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2813..2832
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    3020..3141
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3057..3076)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3111..3130
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3190..3206)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3190..3206)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3203..3225)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3214..3230
                     /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(3238..3244,3245..3262,3263..3268))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3283..3304
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3421..3438)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3592..4180)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3672..3691)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4351..5142,5143..5211))
                     /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     4974..4993
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5212..5316)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5391..5410)
                     /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 tcccaagctg
      661 gctagcgcca ccatgctgca aaagaaactg gaggagctcg aacttcttga tctggcgtct
      721 ctcatactga tggaggaact ttcacaggcc ctggctagta gtttttcagt gtctaccggt
      781 gagaacctgt attttcaggg tatgtctaga ttagataaaa gtaaagtgat taacagcgca
      841 ttagagctgc ttaatgaggt cggaatcgaa ggtttaacaa cccgtaaact cgcccagaag
      901 ctaggtgtag agcagcctac attgtattgg catgtaaaaa ataagcgggc tttgctcgac
      961 gccttagcca ttgagatgtt agataggcac catactcact tttgcccttt agaaggggaa
     1021 agctggcaag attttttacg taataacgct aaaagtttta gatgtgcttt actaagtcat
     1081 cgcgatggag caaaagtaca tttaggtaca cggcctacag aaaaacagta tgaaactctc
     1141 gaaaatcaat tagccttttt atgccaacaa ggtttttcac tagagaatgc attatatgca
     1201 ctcagcgctg tggggcattt tactttaggt tgcgtattgg aagatcaaga gcatcaagtc
     1261 gctaaagaag aaagggaaac acctactact gatagtatgc cgccattatt acgacaagct
     1321 atcgaattat ttgatcacca aggtgcagag ccagccttct tattcggcct tgaattgatc
     1381 atatgcggat tagaaaaaca acttaaatgt gaaagtgggt ccgcgtacag ccgcgcgcgt
     1441 acgaaaaaca attacgggtc taccatcgag ggcctgctcg atctcccgga cgacgacgcc
     1501 cccgaagagg cggggctggc ggctccgcgc ctgtcctttc tccccgcggg acacacgcgc
     1561 agactgtcga cggccccccc gaccgatgtc agcctggggg acgagctcca cttagacggc
     1621 gaggacgtgg cgatggcgca tgccgacgcg ctagacgatt tcgatctgga catgttgggg
     1681 gacggggatt ccccgggtcc gggatttacc ccccacgact ccgcccccta cggcgctctg
     1741 gatatggccg acttcgagtt tgagcagatg tttaccgatg cccttggaat tgacgagtac
     1801 ggtgagaacc tgtattttca gggtggcgcc atggaggaac tttcacaggc cctggctagt
     1861 agtttttcag tgtctcttga tctggcgtct ctcatactgc tgcaaaagaa actggaggag
     1921 ctcgaacttt aggcggccgc tcgagtctag agggcccgtt taaacccgct gatcagcctc
     1981 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     2041 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     2101 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     2161 ttgggaagtc aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     2221 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     2281 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     2341 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     2401 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     2461 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     2521 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     2581 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     2641 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     2701 cagttaggct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     2761 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     2821 cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg
     2881 gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc ggaggttggg
     2941 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     3001 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     3061 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     3121 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     3181 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     3241 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     3301 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     3361 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     3421 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     3481 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     3541 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     3601 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     3661 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     3721 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     3781 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     3841 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     3901 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     3961 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     4021 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     4081 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     4141 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     4201 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     4261 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     4321 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     4381 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     4441 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagatccacg
     4501 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     4561 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     4621 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     4681 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     4741 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     4801 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     4861 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     4921 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     4981 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     5041 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     5101 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     5161 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     5221 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     5281 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     5341 tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc
     5401 tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag
     5461 tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag
     5521 aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt
     5581 t
//
