LOCUS       sequence-356788-        6495 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TEVP-inducible membrane-bound GFP RELEASE construct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6495)
  AUTHORS   Vlahos AE, Kang J, Aldrete CA, Zhu R, Chong LS, Elowitz MB, Gao XJ
  TITLE     Protease-controlled secretion and display of intercellular signals.
  JOURNAL   Nat Commun. 2022 Feb 17;13(1):912. doi: 10.1038/s41467-022-28623-y.
  PUBMED    35177637
REFERENCE   2  (bases 1 to 6495)
  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: "Nat 
            Commun."; date: "2022-02-17"; volume: "13(1):912. doi"; pages: " 
            10.1038/s41467-022-28623-y"
COMMENT     Sequence Label: CMVTO-GFP-ECL-3TM-tevs-KKMP V3
FEATURES             Location/Qualifiers
     source          1..6495
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        180..559
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        560..763
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     714..734
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    765..783
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    786..804
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     814..838
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(993..995,996..998,999..1709)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             1995..2015
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2273..2302
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2358..2375)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2364..2588
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2760..2779)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2911..2958
                     /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             2966..3988
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2966..3988
                     /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    4118..4239
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4155..4174)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4209..4228
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4288..4304)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4288..4304)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4301..4323)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4312..4328
                     /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(4336..4342,4343..4360,4361..4366))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4381..4402
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4519..4536)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4690..5278)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4770..4789)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5449..6240,6241..6309))
                     /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     6072..6091
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6310..6414)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(join(6489..6495,1..13))
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
       61 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
      121 aagaatctgc ttagggttag gcgttttgcg ctgcttcgta cgggccagat atacgcgttg
      181 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
      241 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
      301 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      361 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      421 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      481 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      541 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
      601 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
      661 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
      721 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctcccta tcagtgatag
      781 agatctccct atcagtgata gagatcgtcg acgagctcgt ttagtgaacc gtcagatcgc
      841 ctggagacgc catccacgct gttttgacct ccatagaaga caccgggacc gatccagcct
      901 ccggactcta gcgtttaaac ttaagcttcg ccaccatgca cagctcagca ctgctctgtt
      961 gcctggtcct cctgactggg gtgagggcta gcatggtctc aaagggcgag gaacttttta
     1021 cgggcgttgt tccgatcctc gtcgaattgg acggggacgt taatgggcat aaattctcag
     1081 ttagtggtga gggtgagggg gacgcgacat atgggaaact tacgctgaag ttcatatgta
     1141 ccaccggaaa attgccagtg ccgtggccta ctctcgtcac caccctgaca tatggtgttc
     1201 agtgttttag ccggtatcca gaccatatga aacagcacga tttttttaaa tccgctatgc
     1261 ctgaaggtta cgtacaggag cgaacgatat tttttaaaga cgacggaaac tacaaaacta
     1321 gagccgaggt aaaattcgag ggtgacactc tcgtgaaccg gatcgaactg aagggcatag
     1381 attttaagga ggatggcaat atactgggcc acaagctcga atacaactat aattctcata
     1441 atgtctatat catggcggac aaacagaaaa acggcatcaa ggtaaacttt aaaatccgac
     1501 acaatattga ggacggctct gtccagcttg cggaccatta ccaacagaac acgccgatag
     1561 gggatggtcc agtacttctc cctgataatc actatttgtc tactcagtca gctcttagca
     1621 aggacccaaa cgaaaaacgg gatcacatgg tccttcttga gttcgtcact gcggctggga
     1681 taacactcgg gatggacgaa ctgtataagg gcgccgggac aggagggtct gggggtggca
     1741 ctggcggatc tggaggagac tacaaggacg atgacgacaa cggcggatcg ggggggtctg
     1801 gtggtagtgg cggttcgggg ggaggcacag gtggatccga cgaggtgtgg gtggtgggca
     1861 tgggcatcgt catgtctctc atcgtcctgg ccatcgtgtt tggcaatgtg ctggtcatca
     1921 cagccattgc caagttcgag cgtctgcaga cggtcaccgg gggcggctct ctcgagggcg
     1981 ggggctcggg gctcgaaaac ttgtacttcc agagtggcag tggaggtacc aattacttca
     2041 tcacttcact ggcctgtgct gatctggtca tgggcctggc agtggtgccc tttggggccg
     2101 cccatattct tatgaaaatg tggacttttg gcaacttctg ggatatctac atctgggctc
     2161 ccttggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccctt tactgcaaca
     2221 agtacaaaag cagacgcagt tttattgatg aaaagaaaat gccttagggc ccgaacaaaa
     2281 actcatctca gaagaggatc tgaatagcgc cgtcgaccat catcataatc atcattgagt
     2341 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     2401 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     2461 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     2521 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     2581 ctctatggag atctatcgat gttctgggga tgcggtgggc tctatggctt ctgaggcgga
     2641 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     2701 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     2761 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     2821 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     2881 acttgattag ggtgatggtt cacgtaccta gaagttccta ttccgaagtt cctattctct
     2941 agaaagtata ggaacttcct tggccaaaaa gcctgaactc accgcgacgt ctgtcgagaa
     3001 gtttctgatc gaaaagttcg acagcgtctc cgacctgatg cagctctcgg agggcgaaga
     3061 atctcgtgct ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg taaatagctg
     3121 cgccgatggt ttctacaaag atcgttatgt ttatcggcac tttgcatcgg ccgcgctccc
     3181 gattccggaa gtgcttgaca ttggggaatt cagcgagagc ctgacctatt gcatctcccg
     3241 ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg ctgttctgca
     3301 gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat cttagccaga cgagcgggtt
     3361 cggcccattc ggaccgcaag gaatcggtca atacactaca tggcgtgatt tcatatgcgc
     3421 gattgctgat ccccatgtgt atcactggca aactgtgatg gacgacaccg tcagtgcgtc
     3481 cgtcgcgcag gctctcgatg agctgatgct ttgggccgag gactgccccg aagtccggca
     3541 cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg gacaatggcc gcataacagc
     3601 ggtcattgac tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg ccaacatctt
     3661 cttctggagg ccgtggttgg cttgtatgga gcagcagacg cgctacttcg agcggaggca
     3721 tccggagctt gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg gtcttgacca
     3781 actctatcag agcttggttg acggcaattt cgatgatgca gcttgggcgc agggtcgatg
     3841 cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt acacaaatcg cccgcagaag
     3901 cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc gatagtggaa accgacgccc
     3961 cagcactcgt ccgagggcaa aggaatagca cgtactacga gatttcgatt ccaccgccgc
     4021 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     4081 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa
     4141 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     4201 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
     4261 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     4321 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     4381 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     4441 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     4501 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     4561 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     4621 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     4681 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     4741 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4801 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     4861 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     4921 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     4981 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     5041 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     5101 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     5161 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     5221 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     5281 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     5341 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     5401 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     5461 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     5521 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     5581 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     5641 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     5701 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     5761 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     5821 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     5881 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     5941 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     6001 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     6061 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     6121 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     6181 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     6241 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     6301 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     6361 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     6421 tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtgc
     6481 actctcagta caatc
//
