LOCUS       sequence-224924-        6776 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a combination of proteases.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6776)
  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 6776)
  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-ZscHCVP-TEVPZ
FEATURES             Location/Qualifiers
     source          1..6776
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(189..208)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    340..387
                     /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             395..1417
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             395..1417
                     /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    1547..1668
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1584..1603)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1638..1657
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1717..1733)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1717..1733)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1730..1752)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1741..1757
                     /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(1765..1771,1772..1789,1790..1795))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1810..1831
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1948..1965)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2119..2707)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2199..2218)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2878..3669,3670..3738))
                     /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     3501..3520
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3739..3843)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3918..3937)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4104..4483
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4484..4687
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4638..4658
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    4689..4707
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4710..4728
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     4738..4762
                     /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             5597..5653
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             5654..6361
                     /codon_start=1
                     /product="tobacco etch virus protease (Kapust et al., 
                     2001)"
                     /label=TEV protease
                     /translation="GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNK
                     HLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQ
                     REERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFI
                     VGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPE
                     EPFQPVKEATQLMN"
     CDS             6422..6433
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     CDS             6478..6507
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(6563..6580)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    join(6569..6776,1..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 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
      301 ccccaaaaaa cttgattagg gtgatggttc acgtacctag aagttcctat tccgaagttc
      361 ctattctcta gaaagtatag gaacttcctt ggccaaaaag cctgaactca ccgcgacgtc
      421 tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc gacctgatgc agctctcgga
      481 gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt
      541 aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc
      601 cgcgctcccg attccggaag tgcttgacat tggggaattc agcgagagcc tgacctattg
      661 catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc
      721 tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc ttagccagac
      781 gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt
      841 catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt
      901 cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga
      961 agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg
     1021 cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc
     1081 caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga
     1141 gcggaggcat ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg
     1201 tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca
     1261 gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc
     1321 ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa
     1381 ccgacgcccc agcactcgtc cgagggcaaa ggaatagcac gtactacgag atttcgattc
     1441 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     1501 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
     1561 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     1621 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
     1681 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     1741 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     1801 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     1861 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     1921 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     1981 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     2041 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     2101 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     2161 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     2221 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     2281 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     2341 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     2401 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     2461 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     2521 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     2581 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     2641 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     2701 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     2761 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     2821 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     2881 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     2941 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     3001 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     3061 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     3121 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     3181 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     3241 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     3301 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     3361 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     3421 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     3481 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     3541 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     3601 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     3661 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     3721 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     3781 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     3841 gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc
     3901 ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc
     3961 cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg
     4021 caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt
     4081 cgtacgggcc agatatacgc gttgacattg attattgact agttattaat agtaatcaat
     4141 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
     4201 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
     4261 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
     4321 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
     4381 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
     4441 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
     4501 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
     4561 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
     4621 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
     4681 cagagctctc cctatcagtg atagagatct ccctatcagt gatagagatc gtcgacgagc
     4741 tcgtttagtg aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag
     4801 aagacaccgg gaccgatcca gcctccggac tctagcgttt aaacttaagc tcgccaccat
     4861 gggagagcaa ctcgaaaaga agctgcaagc gttggagaag aagctcgcac aacttgaatg
     4921 gaaaaatcag gcactggaaa agaaactggc tcaaggtggc tcagggagcg gctctgtggt
     4981 gcttgtaggg cgattgctgc tctctggttc tgggtccgcc ccaataacgg cctacgccca
     5041 acagacacgc ggtctgctgg ggtgcattat tacctcactt acgggcagag acaaaaacca
     5101 ggctgaggga gaagttcaga ttgtttcaac ggcggctcag acctttcttg ccacctgtat
     5161 aaacggagta tgctggacag tctaccatgg tgcagggacc cgcacaatag cgagtcccaa
     5221 agggcccgtc atacagatgt atactaacgt cgataaggac ctcgtgggtt ggcccgctcc
     5281 tcaaggcacg cgatccctca caccttgtgc gtgcggcagt tcagacctct atctggtgac
     5341 tcgccatgcc gacgtaatcc ctgttcgccg gaggggtgat agtagaggga gcttgctcag
     5401 tcctaggccg atttcctatc tcaagggctc aagcggcggc ccattgctct gcccggccgg
     5461 ccacgcggta ggaattttcc gggccgcagt ttgcacacgc ggcgtcgcaa aagcagtaga
     5521 ctttatacca gtagagaacc ttgaaacaac gatgaggtcc cctgtattta cagataacag
     5581 ttcccctccg gctgtcgcta ctaatttctc actcctcaaa caagcaggag acgtggaaga
     5641 aaaccctgga cccggggaga gtttgtttaa ggggccaagg gactataacc caatatcatc
     5701 cactatttgc cacctcacta acgagagcga tggacataca acctctctct acgggatagg
     5761 cttcggtcct ttcatcatta ccaataagca tctgtttcgc cgaaacaacg gtactcttct
     5821 ggttcaatct cttcatggtg tcttcaaggt gaaaaacacc actacgcttc agcaacacct
     5881 gattgatggt agggatatga taattatcag aatgccgaaa gatttcccac cttttccaca
     5941 gaagctgaaa ttcagggaac cgcagagaga ggagaggatt tgtttggtaa cgaccaactt
     6001 ccagacgaag agtatgagtt ctatggtgtc cgacactagc tgcacgttcc cctcaagtga
     6061 tgggatattc tggaaacact ggatacagac taaagacgga cagtgtggaa gcccattggt
     6121 ttccacccga gatggtttta tcgtgggtat ccatagcgcc tctaatttca caaacacgaa
     6181 caactatttc acttcagtgc ccaaaaactt tatggagctg ctcacaaacc aagaagcgca
     6241 gcagtgggta agcggttgga gacttaacgc tgactcagtt ctctgggggg ggcacaaagt
     6301 attcatggta aagccagagg agccattcca accagtcaaa gaagccacac aacttatgaa
     6361 cagcggaggc gcgcttaaga aggaacttca agcaaacaaa aaagagttgg cccaactgaa
     6421 gtgggagttg caggcgctca aaaaggagct cgcccagagc tagctcgagg agggcccgaa
     6481 caaaaactca tctcagaaga ggatctgaat agcgccgtcg accatcatca taatcatcat
     6541 tgagtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     6601 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     6661 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     6721 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tgggga
//
