LOCUS       sequence-491571-        8739 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  Munc13-1 protein purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8739)
  AUTHORS   Quade B, Camacho M, Zhao X, Orlando M, Trimbuch T, Xu J, Li W, 
            Nicastro D, Rosenmund C, Rizo J
  TITLE     Membrane bridging by Munc13-1 is crucial for neurotransmitter 
            release.
  JOURNAL   Elife. 2019 Feb 28;8:e42806. doi: 10.7554/eLife.42806.
  PUBMED    30816091
REFERENCE   2  (bases 1 to 8739)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 4, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Elife."; 
            date: "2019-02-28"; volume: "8:e42806. doi"; pages: " 
            10.7554/eLife.42806"
COMMENT     Sequence Label: His-Thrombin-Munc13-1 C1-C2B-MUN-C2C (aa529-17...
FEATURES             Location/Qualifiers
     source          1..8739
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3604..3621
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3674..3692)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3688..3735
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3772..4227
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3859..3878)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4069..4090
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4319..5134)
                     /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             complement(4319..5134)
                     /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     5042..5061
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5126..5145
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5256..5844
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5745..5764
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5998..6015
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6030..6169
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6095..6114)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6255..6277
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6271..6462)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(7271..8353)
                     /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     8315..8334
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8354..8431)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     8637..8656
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg tgccgcgcgg cagccatatg gcgggcatca cctcggcctt ggcctccagc
      181 acgttgaaca atgaagagtt gaaaaaccac gtttacaaga agaccctgca agccttaatc
      241 taccccatct cctgcaccac gccgcacaac ttcgaggtgt ggacggccac cactcccacc
      301 tactgctacg agtgcgaggg gctgctgtgg ggcatcgcgc ggcagggcat gcgctgcacc
      361 gagtgcggcg ttaagtgcca cgagaagtgc caagacctgc tcaacgcgga ctgcctgcag
      421 cgggcggctg agaagagttc taagcatggc gctgaagacc gcacgcagaa catcatcatg
      481 gtgctgaagg accgcatgaa gatccgcgag cgcaacaagc ctgagatctt cgagctgatc
      541 caggaggtct tcgcggtcac caagagcgca cacacacagc agatgaaggc cgtcaagcag
      601 agtgtgctgg atggcacatc caagtggtct gccaaaatta gcatcacggt ggtctgtgcc
      661 cagggcttgc aggccaagga caagacagga tccagtgacc cttatgtcac cgtccaggtt
      721 gggaagacca agaaaaggac aaaaaccatc tacgggaacc ttaacccagt gtgggaagag
      781 aatttccact ttgaatgtca caactcctct gaccggatca aggtgcgtgt gtgggatgaa
      841 gacgatgaca tcaaatcccg tgtgaagcaa aggtttaaga gggagtctga tgacttccta
      901 gggcagacaa tcatcgaggt gcggacgctt agcggcgaga tggatgtgtg gtacaatctg
      961 gacaagagaa cagacaagtc tgctgtgtcg ggcgccattc ggcttcacat cagtgtggag
     1021 atcaaagggg aggagaaggt ggcaccctac catgtccagt acacctgtct gcatgagaac
     1081 ctgttccatt ttgtgacgga tgtacagaac aatggcgtgg tgaagattcc cgatgccaaa
     1141 ggtgacgacg cctggaaggt ttactatgat gagactgccc aggagatcgt ggatgagttt
     1201 gccatgcgct atggcgttga atccatctac caagccatga cccactttgc ctgcctctcc
     1261 tccaagtaca tgtgccctgg ggtacccgct gtcatgagca ccctgcttgc caacatcaac
     1321 gcctactacg cacacaccac cgcctccacc aacgtgtctg cctccgaccg cttcgctgcc
     1381 tccaatttcg ggaaagagcg ctttgtgaaa cttctggacc agctgcacaa ctccctgcgg
     1441 attgacctgt ccatgtaccg gaacaacttc ccggccagca gccccgagcg gctgcaggat
     1501 ctcaagtcca cagtggacct gcttaccagc atcaccttct tccggatgaa ggttcaggaa
     1561 ctgcagagtc cgccacgtgc cagtcaggtg gtgaaggact gcgtgaaggc gtgcctcaac
     1621 tccacctacg agtacatctt caacaactgt catgagctct atggccggga gtaccagaca
     1681 gacccggcca agaaggggga ggtccccccg gaggagcaag gccctagcat caagaacctg
     1741 gatttctggt ccaagctcat caccctcatc gtgtctatca ttgaggagga taagaactcc
     1801 tacacaccct gcctcaatca gtttccccag gagctgaatg tggggaagat cagtgctgag
     1861 gtgatgtgga gcctgtttgc ccaggacatg aagtacgcca tggaggaaca cgacaagcac
     1921 cggctgtgta agagcgcaga ctacatgaac ctgcatttca aggtcaagtg gttgtacaac
     1981 gagtacgtgg ctgaactgcc caccttcaag gaccgcgtgc cggagtaccc tgcgtggttt
     2041 gagcccttcg tcatccagtg gttagatgag aatgaggagg tgtcccggga cttcctgcat
     2101 ggtgcactcg agcgggacaa gaaggatggg ttccagcaga catcggagca cgccctgttc
     2161 tcttgctcgg tagtggacgt cttctcccag ctcaaccaga gctttgagat catcaagaag
     2221 ctggagtgtc ctgaccccca gattgtgggc cactacatgc ggcgctttgc caagaccatt
     2281 agcaatgtgc ttctccagta cgccgacatc gtctccaagg acttcgcttc ctactgctcc
     2341 aaggagaagg agaaagtgcc ctgcatcctc atgaacaaca cacagcagct gcgggtgcag
     2401 ctggagaaga tgttcgaggc gatgggtggg aaggagctgg acgccgaggc cagtggaacc
     2461 ctgaaggagc tgcaggtgaa actcaacaat gtcctggatg aactcagcca cgtgtttgcc
     2521 accagcttcc agccacacat cgaggagtgt gtcagacaaa tgggtgacat cctaagccaa
     2581 gtgaagggca cgggcaacgt gcccgccagt gcctgcagca gcgtggcaca ggacgcagac
     2641 aacgtgctac agcccatcat ggatcttctg gacagcaacc tcaccctgtt tgccaaaatc
     2701 tgtgagaaga cggttctgaa gcgggtgctg aaggagctgt ggaagctggt gatgaacacc
     2761 atggagagga ccattgtcct gccgccatcc aaactgaagg atcacatggt gcgagaagaa
     2821 gccaagagct tgaccccgaa gcagtgtgcc gtggttgaac tggccctgga caccatcaag
     2881 caatacttcc acgcgggggg cgtgggcctc aagaagacct ttctcgagaa aagcccggac
     2941 cttcagtccc tgcgctacgc cctgtcgctc tacacacagg ccaccgacct gctcatcaaa
     3001 accttcgtgc agacgcagtc agcgcagggc tcgggtgtgg aagaccctgt aggtgaagta
     3061 tccgtccacg tggagctgtt cacgcatccg ggaactgggg aacagaaggt cacagtgaag
     3121 gtggtggccg ccaacgacct caagtggcag acttctggca tcttccgtcc gttcattgag
     3181 gtcaacatcg ttggacctca gctcagcgac aagaaacgaa agttcgccac caaatcgaaa
     3241 aacaacagct gggcgcccaa atataacgag agcttccagt tctccctgag cgccgacgcg
     3301 ggacccgagt gctacgagtt gcaggtgtgc gtgaaggact actgcttcgc gcgcgaggac
     3361 cgcacggtgg ggctggcggt gctgcagctg cgggagctgg ctcagcgcgg gagcgccgcg
     3421 tgctggctgc cgctcggccg ccgcatccac atggacgaca cggggctcac agtgctgcgt
     3481 atcctgtcgc agcgcagcaa tgacgaggtg gccaaggagt tcgtcaagct caagtccgac
     3541 acgcgctcgg ccgaggaggg cggtgccgcg cctgcgccct aaaagcttgc ggccgcactc
     3601 gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag
     3661 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
     3721 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
     3781 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     3841 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     3901 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     3961 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     4021 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     4081 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     4141 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     4201 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
     4261 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt
     4321 agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac
     4381 catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata
     4441 ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta
     4501 ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg
     4561 aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc
     4621 cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg
     4681 cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat
     4741 gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt
     4801 cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat
     4861 caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta
     4921 gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca
     4981 actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat
     5041 tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc
     5101 tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt
     5161 aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc gttccactga
     5221 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     5281 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     5341 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     5401 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     5461 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     5521 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     5581 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     5641 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     5701 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     5761 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     5821 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     5881 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     5941 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     6001 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     6061 tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     6121 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     6181 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     6241 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     6301 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
     6361 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     6421 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     6481 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     6541 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     6601 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
     6661 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
     6721 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
     6781 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
     6841 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
     6901 cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt
     6961 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat
     7021 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc
     7081 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt
     7141 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     7201 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     7261 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     7321 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     7381 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     7441 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     7501 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
     7561 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     7621 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     7681 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     7741 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     7801 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     7861 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     7921 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     7981 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     8041 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     8101 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     8161 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     8221 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     8281 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     8341 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     8401 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     8461 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
     8521 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     8581 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     8641 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
     8701 cgtccggcgt agaggatcga gatctcgatc ccgcgaaat
//
