LOCUS       sequence-356883-        6420 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HCVP-inducible tri-transmembrane GFP RELEASE construct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6420)
  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 6420)
  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-26Sfur-3TM-hcvs-KKMP
FEATURES             Location/Qualifiers
     source          1..6420
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        70..449
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        450..653
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     604..624
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    655..673
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    676..694
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     704..728
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(883..885,886..888,889..1599)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             2088..2117
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2173..2190)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2179..2403
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2575..2594)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2726..2773
                     /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             2781..3803
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2781..3803
                     /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    3933..4054
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3970..3989)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4024..4043
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4103..4119)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4103..4119)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4116..4138)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4127..4143
                     /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(4151..4157,4158..4175,4176..4181))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4196..4217
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4334..4351)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4505..5093)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4585..4604)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5264..6055,6056..6124))
                     /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     5887..5906
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6125..6229)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6304..6323)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgta cgggccagat
       61 atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag
      121 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
      181 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
      241 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
      301 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
      361 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
      421 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
      481 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
      541 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
      601 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctcccta
      661 tcagtgatag agatctccct atcagtgata gagatcgtcg acgagctcgt ttagtgaacc
      721 gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga caccgggacc
      781 gatccagcct ccggactcta gcgtttaaac ttaagcttcg ccaccatgca cagctcagca
      841 ctgctctgtt gcctggtcct cctgactggg gtgagggcta gcatggtctc aaagggcgag
      901 gaacttttta cgggcgttgt tccgatcctc gtcgaattgg acggggacgt taatgggcat
      961 aaattctcag ttagtggtga gggtgagggg gacgcgacat atgggaaact tacgctgaag
     1021 ttcatatgta ccaccggaaa attgccagtg ccgtggccta ctctcgtcac caccctgaca
     1081 tatggtgttc agtgttttag ccggtatcca gaccatatga aacagcacga tttttttaaa
     1141 tccgctatgc ctgaaggtta cgtacaggag cgaacgatat tttttaaaga cgacggaaac
     1201 tacaaaacta gagccgaggt aaaattcgag ggtgacactc tcgtgaaccg gatcgaactg
     1261 aagggcatag attttaagga ggatggcaat atactgggcc acaagctcga atacaactat
     1321 aattctcata atgtctatat catggcggac aaacagaaaa acggcatcaa ggtaaacttt
     1381 aaaatccgac acaatattga ggacggctct gtccagcttg cggaccatta ccaacagaac
     1441 acgccgatag gggatggtcc agtacttctc cctgataatc actatttgtc tactcagtca
     1501 gctcttagca aggacccaaa cgaaaaacgg gatcacatgg tccttcttga gttcgtcact
     1561 gcggctggga taacactcgg gatggacgaa ctgtataagg gatcctctgg cagaagcaaa
     1621 agaagcgtca ttgacggctt taccctgacc agcgacgagg tgtgggtggt gggcatgggc
     1681 atcgtcatgt ctctcatcgt cctggccatc gtgtttggca atgtgctggt catcacagcc
     1741 attgccaagt tcgagcgtct gcagacggtc acctctaccg tgagcgagga agctagtgag
     1801 gatgtcgtct gctgctcaat gtcctacaca tggacaggag ccctgaatta cttcatcact
     1861 tcactggcct gtgctgatct ggtcatgggc ctggcagtgg tgccctttgg ggccgcccat
     1921 attcttatga aaatgtggac ttttggcaac ttctgggata tctacatctg ggctcccttg
     1981 gccgggactt gtggggtcct tctcctgtca ctggttatca ccctttactg caacaagtac
     2041 aaaagcagac gcagttttat tgatgaaaag aaaatgcctt agggcccgaa caaaaactca
     2101 tctcagaaga ggatctgaat agcgccgtcg accatcatca taatcatcat tgagtttaaa
     2161 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     2221 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     2281 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     2341 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     2401 tggagatcta tcgatgttct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     2461 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
     2521 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     2581 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     2641 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     2701 attagggtga tggttcacgt acctagaagt tcctattccg aagttcctat tctctagaaa
     2761 gtataggaac ttccttggcc aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc
     2821 tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc gaagaatctc
     2881 gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg
     2941 atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg ctcccgattc
     3001 cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc tcccgccgtg
     3061 cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg
     3121 tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc gggttcggcc
     3181 cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg
     3241 ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg
     3301 cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc cggcacctcg
     3361 tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca
     3421 ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct
     3481 ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg
     3541 agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt gaccaactct
     3601 atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg
     3661 caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg
     3721 ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca
     3781 ctcgtccgag ggcaaaggaa tagcacgtac tacgagattt cgattccacc gccgccttct
     3841 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     3901 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     3961 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     4021 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     4081 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     4141 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     4201 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4261 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4321 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     4381 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4441 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4501 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4561 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     4621 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     4681 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     4741 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     4801 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     4861 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     4921 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     4981 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5041 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5101 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5161 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5221 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5281 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5341 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5401 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     5461 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     5521 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     5581 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     5641 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     5701 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     5761 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     5821 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     5881 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     5941 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     6001 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6061 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6121 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6181 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6241 gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct
     6301 cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
     6361 ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
//
