LOCUS       sequence-407308-        5523 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the SNARE motif of human VAMP3 mutant S44E in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5523)
  AUTHORS   Chen T, Psoma A, Mahajan S, Ter Beest M, Linders P, Franciosa G, 
            Bianchi F, Bogaart GVD, Warner H
  TITLE     Phosphorylation of VAMP3 couples IL-6 Exocytosis to dendritic cell 
            activation.
  JOURNAL   J Cell Sci. 2025 Sep 22:jcs.264139. doi: 10.1242/jcs.264139.
  PUBMED    40977280
REFERENCE   2  (bases 1 to 5523)
  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: "J Cell Sci.
            2025 Sep 22:jcs.264139. doi: 10.1242/jcs.264139."
COMMENT     Sequence Label: hVAMP3(1-77) S44E
FEATURES             Location/Qualifiers
     source          1..5523
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(30..77)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     73..91
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(144..161)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(405..422)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(432..449)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             468..490
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    505..529
                     /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     complement(529..548)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(530..548)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(632..651)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        857..934
                     /gene="lacI"
                     /label=lacI promoter
     CDS             935..2017
                     /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(954..973)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2826..3017
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3011..3033)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3119..3258
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3174..3193
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3273..3290)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3444..4032)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3524..3543)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4143..4162)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4154..4969
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             4154..4969
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4227..4246)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5061..5516)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5198..5219)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5410..5429
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gccaatccgg atatagttcc tcctttcagc aaaaaacccc tcaagacccg tttagaggcc
       61 ccaaggggtt atgctagtta ttgctcagcg gtggcagcag ccaactcagc ttcctttcgg
      121 gctttgttag cagccggatc tcagtggtgg tggtggtggt gctcgagcta tttgcagttt
      181 ttccaccagt atttacgttt cagtttagca gcagaggttt cgaactgaga agcaccagcc
      241 tgcagagcgt cagcacggtc gtccagttct tccagtttct ggtcacgttc cagaactttg
      301 tcaacgttaa cacgcatgat gtcaacaact tcgtcaacct ggttctgggt ctgctgcaga
      361 cgacggttag aaccggtagc agcggtcgga ccggtagaca tatggctgcc gcgcggcacc
      421 aggccgctgc tgtgatgatg atgatgatgg ctgctgccca tggtatatct ccttcttaaa
      481 gttaaacaaa attatttcta gaggggaatt gttatccgct cacaattccc ctatagtgag
      541 tcgtattaat ttcgcgggat cgagatctcg atcctctacg ccggacgcat cgtggccggc
      601 atcaccggcg ccacaggtgc ggttgctggc gcctatatcg ccgacatcac cgatggggaa
      661 gatcgggctc gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc
      721 cccgtggccg ggggactgtt gggcgccatc tccttgcatg caccattcct tgcggcggcg
      781 gtgctcaacg gcctcaacct actactgggc tgcttcctaa tgcaggagtc gcataaggga
      841 gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata
      901 gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt
      961 cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca
     1021 cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc
     1081 caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc
     1141 cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca
     1201 actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc
     1261 ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga
     1321 tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga
     1381 tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact
     1441 gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt
     1501 aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca
     1561 aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac
     1621 catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat
     1681 ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc
     1741 ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat
     1801 caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca
     1861 gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac
     1921 cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     1981 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt
     2041 agctcactca ttaggcaccg ggatctcgac cgatgccctt gagagccttc aacccagtca
     2101 gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta
     2161 tcatgcaact cgtaggacag gtgccggcag cgctctgggt cattttcggc gaggaccgct
     2221 ttcgctggag cgcgacgatg atcggcctgt cgcttgcggt attcggaatc ttgcacgccc
     2281 tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt cggcgagaag caggccatta
     2341 tcgccggcat ggcggcccca cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc
     2401 taggctggcg gggttgcctt actggttagc agaatgaatc accgatacgc gagcgaacgt
     2461 gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg gtcttcggtt
     2521 tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga
     2581 tctgcatcgc aggatgctgc tggctaccct gtggaacacc tacatctgta ttaacgaagc
     2641 gctggcattg accctgagtg atttttctct ggtcccgccg catccatacc gccagttgtt
     2701 taccctcaca acgttccagt aaccgggcat gttcatcatc agtaacccgt atcgtgagca
     2761 tcctctctcg tttcatcggt atcattaccc ccatgaacag aaatccccct tacacggagg
     2821 catcagtgac caaacaggaa aaaaccgccc ttaacatggc ccgctttatc agaagccaga
     2881 cattaacgct tctggagaaa ctcaacgagc tggacgcgga tgaacaggca gacatctgtg
     2941 aatcgcttca cgaccacgct gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg
     3001 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     3061 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg
     3121 cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc
     3181 agagcagatt gtactgagag tgcaccattg cggtgtgaaa taccgcacag atgcgtaagg
     3241 agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     3301 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     3361 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     3421 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     3481 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     3541 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     3601 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     3661 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     3721 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     3781 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     3841 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     3901 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     3961 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     4021 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     4081 aactcacgtt aagggatttt ggtcatgaac aataaaactg tctgcttaca taaacagtaa
     4141 tacaaggggt gttatgagcc atattcaacg ggaaacgtct tgctctaggc cgcgattaaa
     4201 ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc
     4261 aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca
     4321 tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac
     4381 ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt
     4441 actcaccact gcgatccccg ggaaaacagc attccaggta ttagaagaat atcctgattc
     4501 aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt
     4561 ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat
     4621 gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga
     4681 acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca
     4741 tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga
     4801 tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct
     4861 cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc
     4921 tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaag aattaattca
     4981 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     5041 ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     5101 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     5161 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     5221 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     5281 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     5341 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     5401 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     5461 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca
     5521 ttc
//
