LOCUS       sequence-404783-        5523 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the SNARE motif of human VAMP3 mutant S44A 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) S44A
FEATURES             Location/Qualifiers
     source          1..5523
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(401..418)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(428..445)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             464..486
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    501..525
                     /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(525..544)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(526..544)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(628..647)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        853..930
                     /gene="lacI"
                     /label=lacI promoter
     CDS             931..2013
                     /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(950..969)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2822..3013
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3007..3029)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3115..3254
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3170..3189
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3269..3286)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3440..4028)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3520..3539)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4139..4158)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4150..4965
                     /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             4150..4965
                     /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(4223..4242)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5057..5512)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5194..5215)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5406..5425
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagctatttg cagtttttcc
      181 accagtattt acgtttcagt ttagcagcag aggtttcgaa ctgagaagca ccagcctgca
      241 gagcgtcagc acggtcgtcc agttcagcca gtttctggtc acgttccaga actttgtcaa
      301 cgttaacacg catgatgtca acaacttcgt caacctggtt ctgggtctgc tgcagacgac
      361 ggttagaacc ggtagcagcg gtcggaccgg tagacatatg gctgccgcgc ggcaccaggc
      421 cgctgctgtg atgatgatga tgatggctgc tgcccatggt atatctcctt cttaaagtta
      481 aacaaaatta tttctagagg ggaattgtta tccgctcaca attcccctat agtgagtcgt
      541 attaatttcg cgggatcgag atctcgatcc tctacgccgg acgcatcgtg gccggcatca
      601 ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc
      661 gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg
      721 tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc
      781 tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc
      841 gtcgagatcc cggacaccat cgaatggcgc aaaacctttc gcggtatggc atgatagcgc
      901 ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca
      961 gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt
     1021 tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac
     1081 cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt
     1141 ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg
     1201 ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg
     1261 gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac
     1321 caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc
     1381 tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc
     1441 gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt
     1501 tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt
     1561 cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg
     1621 caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg
     1681 ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta
     1741 gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa
     1801 caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc
     1861 caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg
     1921 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     1981 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct
     2041 cactcattag gcaccgggat ctcgaccgat gcccttgaga gccttcaacc cagtcagctc
     2101 cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat
     2161 gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg accgctttcg
     2221 ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc
     2281 tcaagccttc gtcactggtc ccgccaccaa acgtttcggc gagaagcagg ccattatcgc
     2341 cggcatggcg gccccacggg tgcgcatgat cgtgctcctg tcgttgagga cccggctagg
     2401 ctggcggggt tgccttactg gttagcagaa tgaatcaccg atacgcgagc gaacgtgaag
     2461 cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca tgaatggtct tcggtttccg
     2521 tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc accattatgt tccggatctg
     2581 catcgcagga tgctgctggc taccctgtgg aacacctaca tctgtattaa cgaagcgctg
     2641 gcattgaccc tgagtgattt ttctctggtc ccgccgcatc cataccgcca gttgtttacc
     2701 ctcacaacgt tccagtaacc gggcatgttc atcatcagta acccgtatcg tgagcatcct
     2761 ctctcgtttc atcggtatca ttacccccat gaacagaaat cccccttaca cggaggcatc
     2821 agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc tttatcagaa gccagacatt
     2881 aacgcttctg gagaaactca acgagctgga cgcggatgaa caggcagaca tctgtgaatc
     2941 gcttcacgac cacgctgatg agctttaccg cagctgcctc gcgcgtttcg gtgatgacgg
     3001 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
     3061 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc
     3121 catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc ggcatcagag
     3181 cagattgtac tgagagtgca ccattgcggt gtgaaatacc gcacagatgc gtaaggagaa
     3241 aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     3301 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     3361 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     3421 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     3481 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     3541 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     3601 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     3661 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     3721 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     3781 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     3841 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     3901 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     3961 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4021 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4081 cacgttaagg gattttggtc atgaacaata aaactgtctg cttacataaa cagtaataca
     4141 aggggtgtta tgagccatat tcaacgggaa acgtcttgct ctaggccgcg attaaattcc
     4201 aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt
     4261 gcgacaatct atcgattgta tgggaagccc gatgcgccag agttgtttct gaaacatggc
     4321 aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa
     4381 tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc
     4441 accactgcga tccccgggaa aacagcattc caggtattag aagaatatcc tgattcaggt
     4501 gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt
     4561 aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat
     4621 aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc tgttgaacaa
     4681 gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt cactcatggt
     4741 gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt
     4801 ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt
     4861 gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat
     4921 atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaagaatt aattcatgag
     4981 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     5041 ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     5101 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     5161 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     5221 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     5281 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     5341 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     5401 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     5461 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg
     5521 cca
//
