LOCUS       sequence-357122-        6495 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HCVP-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-hcvs-KKMP
FEATURES             Location/Qualifiers
     source          1..6495
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        153..532
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        533..736
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     687..707
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    738..756
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    759..777
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     787..811
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(966..968,969..971,972..1682)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             2246..2275
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2331..2348)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2337..2561
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2733..2752)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2884..2931
                     /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             2939..3961
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2939..3961
                     /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    4091..4212
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4128..4147)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4182..4201
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4261..4277)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4261..4277)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4274..4296)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4285..4301
                     /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(4309..4315,4316..4333,4334..4339))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4354..4375
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4492..4509)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4663..5251)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4743..4762)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5422..6213,6214..6282))
                     /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     6045..6064
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6283..6387)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6462..6481)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct
       61 acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt
      121 gcgctgcttc gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata
      181 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
      241 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
      301 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
      361 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
      421 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
      481 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg
      541 gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
      601 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
      661 atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt
      721 ctatataagc agagctctcc ctatcagtga tagagatctc cctatcagtg atagagatcg
      781 tcgacgagct cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga
      841 cctccataga agacaccggg accgatccag cctccggact ctagcgttta aacttaagct
      901 tcgccaccat gcacagctca gcactgctct gttgcctggt cctcctgact ggggtgaggg
      961 ctagcatggt ctcaaagggc gaggaacttt ttacgggcgt tgttccgatc ctcgtcgaat
     1021 tggacgggga cgttaatggg cataaattct cagttagtgg tgagggtgag ggggacgcga
     1081 catatgggaa acttacgctg aagttcatat gtaccaccgg aaaattgcca gtgccgtggc
     1141 ctactctcgt caccaccctg acatatggtg ttcagtgttt tagccggtat ccagaccata
     1201 tgaaacagca cgattttttt aaatccgcta tgcctgaagg ttacgtacag gagcgaacga
     1261 tattttttaa agacgacgga aactacaaaa ctagagccga ggtaaaattc gagggtgaca
     1321 ctctcgtgaa ccggatcgaa ctgaagggca tagattttaa ggaggatggc aatatactgg
     1381 gccacaagct cgaatacaac tataattctc ataatgtcta tatcatggcg gacaaacaga
     1441 aaaacggcat caaggtaaac tttaaaatcc gacacaatat tgaggacggc tctgtccagc
     1501 ttgcggacca ttaccaacag aacacgccga taggggatgg tccagtactt ctccctgata
     1561 atcactattt gtctactcag tcagctctta gcaaggaccc aaacgaaaaa cgggatcaca
     1621 tggtccttct tgagttcgtc actgcggctg ggataacact cgggatggac gaactgtata
     1681 agggcgccgg gacaggaggg tctgggggtg gcactggcgg atctggagga gactacaagg
     1741 acgatgacga caacggcgga tcgggggggt ctggtggtag tggcggttcg gggggaggca
     1801 caggtggatc cgacgaggtg tgggtggtgg gcatgggcat cgtcatgtct ctcatcgtcc
     1861 tggccatcgt gtttggcaat gtgctggtca tcacagccat tgccaagttc gagcgtctgc
     1921 agacggtcac ctctaccgtg agcgaggaag ctagtgagga tgtcgtctgc tgctcaatgt
     1981 cctacacatg gacaggagcc ctgaattact tcatcacttc actggcctgt gctgatctgg
     2041 tcatgggcct ggcagtggtg ccctttgggg ccgcccatat tcttatgaaa atgtggactt
     2101 ttggcaactt ctgggatatc tacatctggg ctcccttggc cgggacttgt ggggtccttc
     2161 tcctgtcact ggttatcacc ctttactgca acaagtacaa aagcagacgc agttttattg
     2221 atgaaaagaa aatgccttag ggcccgaaca aaaactcatc tcagaagagg atctgaatag
     2281 cgccgtcgac catcatcata atcatcattg agtttaaacc cgctgatcag cctcgactgt
     2341 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     2401 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     2461 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     2521 agacaatagc aggcatgctg gggatgcggt gggctctatg gagatctatc gatgttctgg
     2581 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
     2641 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     2701 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     2761 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     2821 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtac
     2881 ctagaagttc ctattccgaa gttcctattc tctagaaagt ataggaactt ccttggccaa
     2941 aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt
     3001 ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct tcgatgtagg
     3061 agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta
     3121 tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg acattgggga
     3181 attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca cgttgcaaga
     3241 cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca tggatgcgat
     3301 cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc aaggaatcgg
     3361 tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg tgtatcactg
     3421 gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat
     3481 gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa
     3541 caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg aggcgatgtt
     3601 cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt tggcttgtat
     3661 ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat cgccgcggct
     3721 ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg ttgacggcaa
     3781 tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat ccggagccgg
     3841 gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt
     3901 agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg caaaggaata
     3961 gcacgtacta cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     4021 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     4081 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     4141 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     4201 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     4261 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     4321 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     4381 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4441 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     4501 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4561 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4621 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4681 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4741 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4801 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4861 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4921 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4981 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5041 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5101 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5161 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5221 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     5281 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5341 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5401 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5461 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5521 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5581 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5641 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5701 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5761 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5821 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     5881 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     5941 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6001 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6061 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6121 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6181 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6241 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6301 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6361 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga
     6421 cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc
     6481 gcatagttaa gccag
//
