LOCUS       sequence-356787-        7296 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TEVP-inducible GFP-conjugated Kir2.1 RELEASE with KKXX motif.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7296)
  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 7296)
  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-Kir2.1-HA-tevs-KKYL
FEATURES             Location/Qualifiers
     source          1..7296
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        46..425
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        426..629
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     580..600
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    631..649
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    652..670
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     680..704
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(808..810,811..813,814..1524)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     regulatory      1594..1603
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1876..1902
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2863..2883
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2940..2969
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(3025..3042)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3031..3255
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3427..3446)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3578..3625
                     /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             3633..4655
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3633..4655
                     /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    4785..4906
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4822..4841)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4876..4895
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4955..4971)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4955..4971)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4968..4990)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4979..4995
                     /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(5003..5009,5010..5027,5028..5033))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5048..5069
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5186..5203)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5357..5945)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5437..5456)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6116..6907,6908..6976))
                     /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     6739..6758
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6977..7081)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7156..7175)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ggttaggcgt tttgcgctgc ttcgtacggg ccagatatac gcgttgacat tgattattga
       61 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
      121 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
      181 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
      241 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      301 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
      361 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      421 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
      481 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
      541 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
      601 tacggtggga ggtctatata agcagagctc tccctatcag tgatagagat ctccctatca
      661 gtgatagaga tcgtcgacga gctcgtttag tgaaccgtca gatcgcctgg agacgccatc
      721 cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgg actctagcgt
      781 ttaaacttaa gcttcgccac cgctagcatg gtctcaaagg gcgaggaact ttttacgggc
      841 gttgttccga tcctcgtcga attggacggg gacgttaatg ggcataaatt ctcagttagt
      901 ggtgagggtg agggggacgc gacatatggg aaacttacgc tgaagttcat atgtaccacc
      961 ggaaaattgc cagtgccgtg gcctactctc gtcaccaccc tgacatatgg tgttcagtgt
     1021 tttagccggt atccagacca tatgaaacag cacgattttt ttaaatccgc tatgcctgaa
     1081 ggttacgtac aggagcgaac gatatttttt aaagacgacg gaaactacaa aactagagcc
     1141 gaggtaaaat tcgagggtga cactctcgtg aaccggatcg aactgaaggg catagatttt
     1201 aaggaggatg gcaatatact gggccacaag ctcgaataca actataattc tcataatgtc
     1261 tatatcatgg cggacaaaca gaaaaacggc atcaaggtaa actttaaaat ccgacacaat
     1321 attgaggacg gctctgtcca gcttgcggac cattaccaac agaacacgcc gataggggat
     1381 ggtccagtac ttctccctga taatcactat ttgtctactc agtcagctct tagcaaggac
     1441 ccaaacgaaa aacgggatca catggtcctt cttgagttcg tcactgcggc tgggataaca
     1501 ctcgggatgg acgaactgta taagggcgcc atgggcagtg tgcgaaccaa ccgctacagc
     1561 atcgtctctt cagaagaaga cggtatgaag ttggccacca tggcagttgc aaatggcttt
     1621 gggaacggga agagtaaagt ccacacccga caacagtgca ggagccgctt tgtgaagaaa
     1681 gatggccact gtaatgttca gttcatcaat gtgggtgaga aggggcaacg gtatctcgca
     1741 gacatcttca ccacgtgtgt ggacattcgc tggcggtgga tgctggttat cttctgcctg
     1801 gctttcgtcc tgtcatggct gttttttggc tgtgtgtttt ggttgatagc tctgctccat
     1861 ggggacctgg atgcataccc atacgatgtt ccagattacg cttccaaaga gggcaaggct
     1921 tgtgtgtccg aggtcaacag cttcacggct gccttcctct tctccattga gacccagaca
     1981 accataggct atggtttcag atgtgtcacg gatgaatgcc caattgctgt tttcatggtg
     2041 gtgttccagt caatcgtggg ctgcatcata gatgctttca tcattggcgc agtcatggcc
     2101 aagatggcaa agccaaagaa gagaaacgag actcttgtct tcagtcacaa tgccgtgatt
     2161 gccatgagag acggcaagct gtgtttgatg tggcgagtgg gcaatcttcg gaaaagccac
     2221 ttggtggaag ctcatgttcg agcacagctc ctcaaatcca gaattacttc tgaaggggag
     2281 tatatccctc tggatcaaat agacatcaat gttgggtttg acagtggaat agatcgtata
     2341 tttctggtgt ccccaatcac tatagtccat gaaatagatg aagacagtcc tttatatgat
     2401 ttgagtaaac aggacattga caacgcagac tttgaaatcg tggtcatact ggaaggcatg
     2461 gtggaagcca ctgccatgac gacacagtgc cgtagctctt atctagcaaa tgaaatcctg
     2521 tggggccacc gctatgagcc tgtgctcttt gaagagaagc actactacaa agtggactat
     2581 tccaggttcc acaaaactta cgaagtcccc aacactcccc tttgtagtgc cagagactta
     2641 gcagaaaaga aatatatcct ctcaaatgca aattcatttt gctatgaaaa tgaagttgcc
     2701 ctcacaagca aagaggaaga cgacagtgaa aatggagttc cagaaagcac tagtacggac
     2761 acgccccctg acatagacct tcacaaccag gcaagtgtac ctctagagcc caggccctta
     2821 cggcgagagt cggagatact cgagggcggg ggctcggggc tcgaaaactt gtacttccag
     2881 agtggcagtg gaggtaccct ggcctccagc ttgactttca agaaatacct gtagggcccg
     2941 aacaaaaact catctcagaa gaggatctga atagcgccgt cgaccatcat cataatcatc
     3001 attgagttta aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     3061 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     3121 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     3181 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     3241 cggtgggctc tatggagatc tatcgatgtt ctggggatgc ggtgggctct atggcttctg
     3301 aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat
     3361 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     3421 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     3481 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     3541 ccaaaaaact tgattagggt gatggttcac gtacctagaa gttcctattc cgaagttcct
     3601 attctctaga aagtatagga acttccttgg ccaaaaagcc tgaactcacc gcgacgtctg
     3661 tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag ctctcggagg
     3721 gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa
     3781 atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatcggccg
     3841 cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg acctattgca
     3901 tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg
     3961 ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc ggccgatctt agccagacga
     4021 gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca
     4081 tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca
     4141 gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag
     4201 tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca
     4261 taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca
     4321 acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc
     4381 ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc cgcattggtc
     4441 ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg
     4501 gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc
     4561 gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc
     4621 gacgccccag cactcgtccg agggcaaagg aatagcacgt actacgagat ttcgattcca
     4681 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     4741 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     4801 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4861 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     4921 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     4981 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     5041 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     5101 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5161 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     5221 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5281 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5341 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     5401 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5461 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5521 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     5581 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5641 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5701 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5761 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5821 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5881 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5941 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     6001 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     6061 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     6121 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     6181 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     6241 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     6301 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6361 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6421 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6481 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6541 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6601 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6661 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6721 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6781 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6841 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6901 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6961 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     7021 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     7081 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
     7141 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
     7201 tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca
     7261 aggcttgacc gacaattgca tgaagaatct gcttag
//
