LOCUS       sequence-219754-        6194 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses an engineered TVMV protease.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6194)
  AUTHORS   Gao XJ, Chong LS, Kim MS, Elowitz MB
  TITLE     Programmable protein circuits in living cells.
  JOURNAL   Science. 2018 Sep 21;361(6408):1252-1258. doi: 
            10.1126/science.aat5062.
  PUBMED    30237357
REFERENCE   2  (bases 1 to 6194)
  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: "10.1126/science"; 
            journalName: "Science"; date: "2018-09-21- 21"; volume: "361"; 
            issue: "6408"; pages: "1252-1258"
COMMENT     Sequence Label: CMV-TO-nTVMVPhcZhccTVMVPZ
FEATURES             Location/Qualifiers
     source          1..6194
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(189..208)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    339..386
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             394..1416
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             394..1416
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    1546..1667
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1583..1602)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1637..1656
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1716..1732)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1716..1732)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1729..1751)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1740..1756
                     /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(1764..1770,1771..1788,1789..1794))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1809..1830
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1947..1964)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2118..2706)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2198..2217)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2877..3668,3669..3737))
                     /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     3500..3519
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3738..3842)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3917..3936)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4103..4482
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4483..4686
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4637..4657
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    4688..4706
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4709..4727
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     4737..4761
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      4853..4862
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5333..5344
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     CDS             5840..5851
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     CDS             5896..5925
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5941..5958
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5981..5998)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    join(5987..6194,1..17)
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 tgcggtgggc tctatggaga tctatcgatg ttctggggat gcggtgggct ctatggcttc
       61 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
      121 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
      181 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
      241 tcaagctcta aatcgggggt ccctttaggg ttccgattta gtgctttacg gcacctcgac
      301 cccaaaaaac ttgattaggg tgatggttca cgtacctaga agttcctatt ccgaagttcc
      361 tattctctag aaagtatagg aacttccttg gccaaaaagc ctgaactcac cgcgacgtct
      421 gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag
      481 ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta
      541 aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc
      601 gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc
      661 atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct
      721 gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg
      781 agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc
      841 atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc
      901 agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa
      961 gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc
     1021 ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc
     1081 aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag
     1141 cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt
     1201 cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag
     1261 ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc
     1321 cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac
     1381 cgacgcccca gcactcgtcc gagggcaaag gaatagcacg tactacgaga tttcgattcc
     1441 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     1501 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     1561 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     1621 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     1681 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     1741 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     1801 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     1861 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     1921 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     1981 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     2041 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     2101 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     2161 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     2221 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     2281 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     2341 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     2401 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     2461 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     2521 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
     2581 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     2641 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     2701 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     2761 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     2821 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     2881 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     2941 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     3001 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     3061 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     3121 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     3181 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     3241 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     3301 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     3361 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     3421 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     3481 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     3541 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     3601 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     3661 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     3721 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     3781 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     3841 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc
     3901 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc
     3961 ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc
     4021 aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc
     4081 gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt
     4141 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
     4201 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
     4261 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     4321 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
     4381 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
     4441 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
     4501 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
     4561 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
     4621 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
     4681 agagctctcc ctatcagtga tagagatctc cctatcagtg atagagatcg tcgacgagct
     4741 cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga
     4801 agacaccggg accgatccag cctccggact ctagcgttta aacttaagct tcgccaccat
     4861 gggatcctca aaagcactcc tgaagggagt ccgcgacttt aatcctatat cagcgtgtgt
     4921 atgtttgctt gaaaactcct ctgatgggca ttcagaaagg ctttttggta ttgggtttgg
     4981 tccctacata atagcaaacc aacacctgtt tcgacgcaat aacggcgagc ttacgattaa
     5041 gacgatgcac ggggaattta aagtgaaaaa cagcacacaa ttgcaaatga aacccgtcga
     5101 agggagagac atcattgtaa taaagatggc aaaggatttc ccaccctttc cgcagaaatt
     5161 gaaattccgg caacctacta taaaggatcg agtttgtatg gtttctacga actttcagca
     5221 aggctctggc agcagcgacg agatggagga gtgtagtcag caccttggcg gaggtggcag
     5281 cgcgcttaag aaggaacttc aagcaaacaa aaaagagttg gcccaactga agtgggagtt
     5341 gcaggcgctc aaaaaggagc tcgcccaggg tggcggagga tctgatgaga tggaagagtg
     5401 ctcccaacat ttgggagggt cagggagcaa atcagtctcc tccctcgtgt ccgaatcctc
     5461 tcacatcgtt cacaaggaag ataccagttt ctggcagcat tggattacca caaaagacgg
     5521 gcagtgcggc tcaccgctgg tgtctatcat tgacgggaac attctgggaa tccactccct
     5581 gacacacacc actaacggct caaactattt cgttgagttc ccagagaagt tcgtagctac
     5641 atatctggac gctgcggacg gctggtgcaa gaactggaaa ttcaacgcgg acaagatttc
     5701 atggggtagt ttcacgcttg tcgaagacgc gcccgaggac gactttatgg cgaaaaaaac
     5761 cgtggctgct attatggata gcggaggcgc gcttaagaag gaacttcaag caaacaaaaa
     5821 agagttggcc caactgaagt gggagttgca ggcgctcaaa aaggagctcg cccagagcta
     5881 gctcgaggag ggcccgaaca aaaactcatc tcagaagagg atctgaatag cgccgtcgac
     5941 catcatcatc atcatcattg agtttaaacc cgctgatcag cctcgactgt gccttctagt
     6001 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     6061 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     6121 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     6181 aggcatgctg ggga
//
