LOCUS       sequence-328408-        7845 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 7845)
  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 7845)
  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..7845
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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             42..64
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     promoter        784..802
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    803..827
                     /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             1749..1793
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     terminator      1845..1892
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1929..2384
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(join(2499..3290,3291..3359))
                     /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     3122..3141
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3360..3452)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3533..4121
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4022..4041
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4275..4292
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4307..4449
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4375..4394)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4535..4557
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4551..4742)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5314..6396)
                     /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     6358..6377
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6397..6474)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6680..6699
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        6783..6801
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6802..6826
                     /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             6841..6863
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6885..6902
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6924..6944
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     terminator      7281..7328
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      7365..7793
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        join(7829..7845,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatatacc atggctagca tggccgaaga cgcggatatg cgcaatgagc tggaggagat
      121 gcagaggagg gctgaccagc tggctgatga gtccctggaa agcacccgtc gcatgctgca
      181 gctggttgaa gagagtaaag atgctggcat caggactttg gttatgttgg atgagcaagg
      241 cgaacaactc gatcgtgtcg aagaaggcat gaaccatatc aaccaagaca tgaaggaggc
      301 cgagaaaaat ttaaaagatt taggcaaatg ctgtggcctt ttcatatgtc cttgtaacaa
      361 gcttaaatcc agtgatgctt acaaaaaagc ctggggcaat aatcaggatg gagtagtggc
      421 cagccagccc gcccgtgtgg tggatgaacg ggagcagatg gccatcagtg gtggcttcat
      481 ccgcagggta acaaatgatg cccgggaaaa tgagatggat gagaacctgg agcaggtgag
      541 cggcatcatc ggaaacctcc gccatatggc actagacatg ggcaatgaga ttgacaccca
      601 gaatcgccag atcgacagga tcatggagaa ggctgattcc aacaaaacca gaattgatga
      661 agccaaccaa cgtgcaacaa agatgctggg cagtgggtaa tagtgactcg acaagcttgc
      721 ggccgcataa tgcttaagtc gaacagaaag taatcgtatt gtacacggcc gcataatcga
      781 aattaatacg actcactata ggggaattgt gagcggataa caattcccca tcttagtata
      841 ttagttaagt ataagaagga gatatacata tgaaggaccg aacccaggag ctccgcacgg
      901 ccaaggacag cgatgacgac gatgatgtca ctgtcactgt ggaccgagac cgcttcatgg
      961 atgagttctt tgaacaggtg gaagagatcc ggggctttat tgacaagatt gctgaaaacg
     1021 tggaggaggt gaagaggaaa cacagcgcca tcctggcctc cccgaacccc gatgagaaga
     1081 ccaaggagga actggaagag ctcatgtcgg acattaagaa gacagcgaac aaagttcgct
     1141 ccaagctaaa gagcatcgag cagagcatcg agcaggagga aggtctgaac cgctcgtcgg
     1201 cggacctgag gatccggaag acgcagcatt ccacgctgtc ccgaaagttt gtggaggtca
     1261 tgtccgagta caacgccact cagtcagact accgagaacg ctccaaaggg cgcatccaga
     1321 ggcagctgga gatcactggc cggaccacga ccagtgagga gttggaagac atgctggaga
     1381 gtgggaatcc cgccatcttt gcctctggga tcatcatgga ctccagcatc tcgaagcagg
     1441 ccctcagtga gatcgagacc aggcacagtg agatcatcaa gttggagaac agcatccggg
     1501 agctacacga tatgttcatg gacatggcca tgctggtgga gagccagggg gagatgattg
     1561 acaggatcga gtacaatgtg gaacacgctg tggactacgt ggagagggcc gtgtgtgaca
     1621 ccaagtgcgc cgtcaagtac cagagcaagg cacgcaggaa gaagtagatg atcatcatta
     1681 gcagtgtgat tctgggcatc atcatcgcct ccaccatcgg gggcatcttt ggatagctcg
     1741 agtctggtaa agaaaccgct gctgcgaaat ttgaacgcca gcacatggac tcgtctacta
     1801 gcgcagctta attaacctag gctgctgcca ccgctgagca ataactagca taaccccttg
     1861 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg
     1921 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     1981 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     2041 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     2101 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     2161 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     2221 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     2281 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     2341 aaaatttaac gcgaatttta acaaaatatt aacgtttaca atttctggcg gcacgatggc
     2401 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     2461 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     2521 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     2581 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     2641 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     2701 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     2761 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     2821 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     2881 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     2941 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     3001 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     3061 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     3121 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     3181 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     3241 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     3301 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     3361 ctcttccttt ttcaatcatg attgaagcat ttatcagggt tattgtctca tgagcggata
     3421 catatttgaa tgtatttaga aaaataaaca aataggtcat gaccaaaatc ccttaacgtg
     3481 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     3541 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     3601 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     3661 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     3721 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     3781 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     3841 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     3901 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     3961 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     4021 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     4081 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     4141 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     4201 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     4261 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     4321 ttctccttac gcatctgtgc ggtatttcac accgcatata tggtgcactc tcagtacaat
     4381 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     4441 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     4501 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     4561 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     4621 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     4681 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     4741 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     4801 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     4861 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     4921 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     4981 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     5041 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     5101 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     5161 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc tagtcatgcc ccgcgcccac
     5221 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     5281 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     5341 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     5401 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     5461 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     5521 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     5581 tcccactacc gagatgtccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     5641 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     5701 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     5761 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     5821 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     5881 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     5941 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     6001 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     6061 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     6121 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     6181 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     6241 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     6301 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     6361 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     6421 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     6481 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     6541 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     6601 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
     6661 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
     6721 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa
     6781 attaatacga ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat
     6841 tttgtttaac tttaagaagg agatatacat atatgtcgta ctaccatcac catcaccatc
     6901 acgattacga tatcccaact agtgaaaacc tgtattttca gggcgctagc catatgtcgg
     6961 ctaccgctgc caccgtcccg cctgccgccc cggccggcga gggtggcccc cctgcacctc
     7021 ctccaaatct taccagcaac aggagactgc agcagaccca ggcccaggtg gatgaggtgg
     7081 tggacatcat gagggtgaat gtggacaagg tcctggagcg agaccagaag ctatcggaac
     7141 tggatgatcg cgcagatgcc ctccaggcag gggcctccca gtttgaaaca agtgcagcca
     7201 agctcaagcg caaatactgg tggaaaaacc tcaagatgat gtagctcgag ctcccgggat
     7261 ccgcggccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     7321 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     7381 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     7441 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     7501 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     7561 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     7621 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     7681 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     7741 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     7801 aatattaacg atctcgatcc cgcgaaatta atacgactca ctata
//
