LOCUS       sequence-333023-        8170 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Syntaxin1-265 C145S, S249C, K253C ; SNAP25 ; 
            6xHis-TEV-Synaptobrevin1-96.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8170)
  AUTHORS   Cipriano DJ, Jung J, Vivona S, Fenn TD, Brunger AT, Bryant Z
  TITLE     Processive ATP-driven substrate disassembly by the 
            N-ethylmaleimide-sensitive factor (NSF) molecular machine.
  JOURNAL   J Biol Chem. 2013 Aug 9;288(32):23436-45. doi: 
            10.1074/jbc.M113.476705. Epub 2013 Jun 17.
  PUBMED    23775070
REFERENCE   2  (bases 1 to 8170)
  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: 
            "10.1074/jbc.M113.476705"; journalName: "J Biol Chem"; date: 
            "2013-08-9- 9"; volume: "288"; issue: "32"; pages: "23436-45"
COMMENT     Sequence Label: pDC146
FEATURES             Location/Qualifiers
     source          1..8170
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        272..290
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    291..315
                     /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)."
     RBS             330..352
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     promoter        1072..1090
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1091..1115
                     /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             2037..2081
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     terminator      2133..2180
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2217..2672
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(join(2787..3578,3579..3647))
                     /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     3410..3429
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3648..3740)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3821..4409
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4310..4329
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4563..4580
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4595..4734
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4660..4679)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4820..4842
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4836..5027)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5599..6681)
                     /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     6643..6662
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6682..6759)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6965..6984
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        7068..7086
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7087..7111
                     /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)."
     RBS             7126..7148
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             7170..7187
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             7209..7229
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     terminator      7566..7613
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      7650..8105
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        join(8131..8170,1..65)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
       61 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
      121 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
      181 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
      241 gccccgaaga acgatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga
      301 gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga gatataccat
      361 ggctagcatg gccgaagacg cggatatgcg caatgagctg gaggagatgc agaggagggc
      421 tgaccagctg gctgatgagt ccctggaaag cacccgtcgc atgctgcagc tggttgaaga
      481 gagtaaagat gctggcatca ggactttggt tatgttggat gagcaaggcg aacaactcga
      541 tcgtgtcgaa gaaggcatga accatatcaa ccaagacatg aaggaggccg agaaaaattt
      601 aaaagattta ggcaaatgct gtggcctttt catatgtcct tgtaacaagc ttaaatccag
      661 tgatgcttac aaaaaagcct ggggcaataa tcaggatgga gtagtggcca gccagcccgc
      721 ccgtgtggtg gatgaacggg agcagatggc catcagtggt ggcttcatcc gcagggtaac
      781 aaatgatgcc cgggaaaatg agatggatga gaacctggag caggtgagcg gcatcatcgg
      841 aaacctccgc catatggcac tagacatggg caatgagatt gacacccaga atcgccagat
      901 cgacaggatc atggagaagg ctgattccaa caaaaccaga attgatgaag ccaaccaacg
      961 tgcaacaaag atgctgggca gtgggtaata gtgactcgac aagcttgcgg ccgcataatg
     1021 cttaagtcga acagaaagta atcgtattgt acacggccgc ataatcgaaa ttaatacgac
     1081 tcactatagg ggaattgtga gcggataaca attccccatc ttagtatatt agttaagtat
     1141 aagaaggaga tatacatatg aaggaccgaa cccaggagct ccgcacggcc aaggacagcg
     1201 atgacgacga tgatgtcact gtcactgtgg accgagaccg cttcatggat gagttctttg
     1261 aacaggtgga agagatccgg ggctttattg acaagattgc tgaaaacgtg gaggaggtga
     1321 agaggaaaca cagcgccatc ctggcctccc cgaaccccga tgagaagacc aaggaggaac
     1381 tggaagagct catgtcggac attaagaaga cagcgaacaa agttcgctcc aagctaaaga
     1441 gcatcgagca gagcatcgag caggaggaag gtctgaaccg ctcgtcggcg gacctgagga
     1501 tccggaagac gcagcattcc acgctgtccc gaaagtttgt ggaggtcatg tccgagtaca
     1561 acgccactca gtcagactac cgagaacgct ccaaagggcg catccagagg cagctggaga
     1621 tcactggccg gaccacgacc agtgaggagt tggaagacat gctggagagt gggaatcccg
     1681 ccatctttgc ctctgggatc atcatggact ccagcatctc gaagcaggcc ctcagtgaga
     1741 tcgagaccag gcacagtgag atcatcaagt tggagaacag catccgggag ctacacgata
     1801 tgttcatgga catggccatg ctggtggaga gccaggggga gatgattgac aggatcgagt
     1861 acaatgtgga acacgctgtg gactacgtgg agagggccgt gtgtgacacc aagtgcgccg
     1921 tcaagtacca gagcaaggca cgcaggaaga agtagatgat catcattagc agtgtgattc
     1981 tgggcatcat catcgcctcc accatcgggg gcatctttgg atagctcgag tctggtaaag
     2041 aaaccgctgc tgcgaaattt gaacgccagc acatggactc gtctactagc gcagcttaat
     2101 taacctaggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     2161 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
     2221 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     2281 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     2341 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     2401 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     2461 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     2521 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     2581 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     2641 gaattttaac aaaatattaa cgtttacaat ttctggcggc acgatggcat gagattatca
     2701 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     2761 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     2821 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     2881 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     2941 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     3001 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     3061 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     3121 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     3181 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     3241 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     3301 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     3361 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     3421 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     3481 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     3541 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     3601 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     3661 caatcatgat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     3721 tatttagaaa aataaacaaa taggtcatga ccaaaatccc ttaacgtgag ttttcgttcc
     3781 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     3841 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     3901 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     3961 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     4021 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     4081 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     4141 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     4201 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     4261 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     4321 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     4381 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     4441 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     4501 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     4561 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     4621 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     4681 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     4741 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     4801 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     4861 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     4921 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     4981 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     5041 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     5101 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     5161 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     5221 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     5281 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     5341 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     5401 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     5461 ctcaacgaca ggagcacgat catgctagtc atgccccgcg cccaccggaa ggagctgact
     5521 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     5581 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     5641 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     5701 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     5761 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     5821 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     5881 gtccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     5941 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     6001 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     6061 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     6121 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     6181 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     6241 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     6301 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     6361 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     6421 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     6481 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     6541 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     6601 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     6661 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     6721 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     6781 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     6841 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     6901 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
     6961 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     7021 ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac
     7081 tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa
     7141 gaaggagata tacatatatg tcgtactacc atcaccatca ccatcacgat tacgatatcc
     7201 caactagtga aaacctgtat tttcagggcg ctagccatat gtcggctacc gctgccaccg
     7261 tcccgcctgc cgccccggcc ggcgagggtg gcccccctgc acctcctcca aatcttacca
     7321 gcaacaggag actgcagcag acccaggccc aggtggatga ggtggtggac atcatgaggg
     7381 tgaatgtgga caaggtcctg gagcgagacc agaagctatc ggaactggat gatcgcgcag
     7441 atgccctcca ggcaggggcc tcccagtttg aaacaagtgc agccaagctc aagcgcaaat
     7501 actggtggaa aaacctcaag atgatgtagc tcgagctccc gggatccgcg gccgctgagc
     7561 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     7621 gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     7681 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     7741 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     7801 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     7861 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     7921 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     7981 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     8041 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     8101 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     8161 atacattcaa
//
