LOCUS       sequence-387506-        5363 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses VAMP8 in E. Coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5363)
  AUTHORS   Lai Y, Fois G, Flores JR, Tuvim MJ, Zhou Q, Yang K, Leitz J, Peters 
            J, Zhang Y, Pfuetzner RA, Esquivies L, Jones P, Frick M, Dickey BF, 
            Brunger AT
  TITLE     Inhibition of calcium-triggered secretion by hydrocarbon-stapled 
            peptides.
  JOURNAL   Nature. 2022 Mar;603(7903):949-956. doi: 10.1038/s41586-022-04543-1.
            Epub 2022 Mar 23.
  PUBMED    35322233
REFERENCE   2  (bases 1 to 5363)
  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: 
            "10.1038/s41586-022-04543-1"; journalName: "Nature"; date: 
            "2022-03"; volume: "603"; issue: "7903"; pages: "949-956"
COMMENT     Sequence Label: VAMP8
FEATURES             Location/Qualifiers
     source          1..5363
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(420..425,426..443,444..450)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    458..474
                     /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)."
     primer_bind     482..498
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     482..498
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             494..1021
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(514..531)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(514..530)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(523..545)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(1123..1128,1129..1144,1145..1151)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1159..1175
                     /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)."
     CDS             1198..1851
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1809..1831
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             1858..1875
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1876..1896
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1897..1962
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2353..2375)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2520..2538)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2606..2710
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2711..2779,2780..3571)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2929..2948)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3742..4330
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4231..4250
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4484..4501
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4574..4651
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(4652..5363,1..371)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4671..4690)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta
       61 ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag
      121 acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc
      181 aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc
      241 tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct
      301 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
      361 gcgggcagtg agcgcaacgc aattaatgta agttagctca ctcattaggc accccaggct
      421 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
      481 acaggaaaca gctatgacca tgattacgga ttcactggcc gtcgttttac aacgtcgtga
      541 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
      601 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
      661 tggcgaatgg cgctttgcct ggtttccggc accagaagcg gtgccggaaa gctggctgga
      721 gtgcgatctt cctgaggccg atactgtcgt cgtcccctca aactggcaga tgcacggtta
      781 cgatgcgccc atctacacca acgtgaccta tcccattacg gtcaatccgc cgtttgttcc
      841 cacggagaat ccgacgggtt gttactcgct cacatttaat gttgatgaaa gctggctaca
      901 ggaaggccag acgcgaatta tttttgatgg cgttggaatt agcttatcga ctgcacggtg
      961 caccaatgct tctggcgtca ggcagccatc ggaagctgtg gtatggctgt gcaggtcgta
     1021 aatcactgca taattcgtgt cgctcaaggc gcactcccgt tctggataat gttttttgcg
     1081 ccgacatcat aacggttctg gcaaatattc tgaaatgagc tgttgacaat taatcatcgg
     1141 ctcgtataat gtgtggaatt gtgagcggat aacaatttca cacaggaaac agtattcatg
     1201 tcccctatac taggttattg gaaaattaag ggccttgtgc aacccactcg acttcttttg
     1261 gaatatcttg aagaaaaata tgaagagcat ttgtatgagc gcgatgaagg tgataaatgg
     1321 cgaaacaaaa agtttgaatt gggtttggag tttcccaatc ttccttatta tattgatggt
     1381 gatgttaaat taacacagtc tatggccatc atacgttata tagctgacaa gcacaacatg
     1441 ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc ttgaaggagc ggttttggat
     1501 attagatacg gtgtttcgag aattgcatat agtaaagact ttgaaactct caaagttgat
     1561 tttcttagca agctacctga aatgctgaaa atgttcgaag atcgtttatg tcataaaaca
     1621 tatttaaatg gtgatcatgt aacccatcct gacttcatgt tgtatgacgc tcttgatgtt
     1681 gttttataca tggacccaat gtgcctggat gcgttcccaa aattagtttg ttttaaaaaa
     1741 cgtattgaag ctatcccaca aattgataag tacttgaaat ccagcaagta tatagcatgg
     1801 cctttgcagg gctggcaagc cacgtttggt ggtggcgacc atcctccaaa atcggatctg
     1861 gttccgcgtg gatctgaaaa cttatatttt cagggcgact acaaagacca tgacggtgat
     1921 tataaagatc atgacatcga ctacaaggat gacgatgaca agggatcccc ggaattcccg
     1981 ggtcgactca tggaggaagc cagtgaaggt ggaggaaatg atcgtgtgcg gaacctgcaa
     2041 agtgaggtgg agggagttaa gaatattatg acccagaatg tggagcggat cctggcccgg
     2101 ggggaaaact tggaacatct ccgcaacaag acagaggatc tggaagccac atctgagcac
     2161 ttcaagacga catcgcagaa ggtggctcgg aaattctggt ggaagaacgt gaagatgatt
     2221 gtccttatct gcgtgattgt ttttatcatc atcctcttca ttgtgctctt tgccactggt
     2281 gccttctctt aagcggccgc atcgtgactg actgacgatc tgcctcgcgc gtttcggtga
     2341 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
     2401 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
     2461 cgcagccatg acccagtcac gtagcgatag cggagtgtat aattcttgaa gacgaaaggg
     2521 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     2581 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2641 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     2701 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     2761 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     2821 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     2881 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     2941 ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     3001 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     3061 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     3121 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     3181 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     3241 caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     3301 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     3361 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     3421 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     3481 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     3541 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     3601 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     3661 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     3721 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3781 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3841 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     3901 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     3961 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     4021 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     4081 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     4141 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     4201 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     4261 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     4321 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4381 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     4441 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     4501 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     4561 ccgcataaat tccgacacca tcgaatggcg caaaaccttt cgcggtatgg catgatagcg
     4621 cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc
     4681 agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt
     4741 ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa
     4801 ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag
     4861 tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact
     4921 gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc
     4981 ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga
     5041 ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt
     5101 ctctgaccag acacccatca acagtattat tttctcccat gaagacggta cgcgactggg
     5161 cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag
     5221 ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat
     5281 tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat
     5341 gcaaatgctg aatgagggca tcg
//
