LOCUS       sequence-486130-        8736 bp    DNA     circular SYN 16-SEP-2025
DEFINITION  Munc13-1 protein purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8736)
  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 8736)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 16, 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 R1598E F1...
FEATURES             Location/Qualifiers
     source          1..8736
                     /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             124..141
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3601..3618
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3671..3689)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3685..3732
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3769..4224
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3856..3875)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4066..4087
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4316..5131)
                     /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(4316..5131)
                     /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     5039..5058
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5123..5142
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5253..5841
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5742..5761
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5995..6012
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6027..6166
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6092..6111)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6252..6274
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6268..6459)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(7268..8350)
                     /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     8312..8331
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8351..8428)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     8634..8653
                     /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 tcacagcagc
      121 ggcctggtgc cgcgcggcag ccatatggcg ggcatcacct cggccttggc ctccagcacg
      181 ttgaacaatg aagagttgaa aaaccacgtt tacaagaaga ccctgcaagc cttaatctac
      241 cccatctcct gcaccacgcc gcacaacttc gaggtgtgga cggccaccac tcccacctac
      301 tgctacgagt gcgaggggct gctgtggggc atcgcgcggc agggcatgcg ctgcaccgag
      361 tgcggcgtta agtgccacga gaagtgccaa gacctgctca acgcggactg cctgcagcgg
      421 gcggctgaga agagttctaa gcatggcgct gaagaccgca cgcagaacat catcatggtg
      481 ctgaaggacc gcatgaagat ccgcgagcgc aacaagcctg agatcttcga gctgatccag
      541 gaggtcttcg cggtcaccaa gagcgcacac acacagcaga tgaaggccgt caagcagagt
      601 gtgctggatg gcacatccaa gtggtctgcc aaaattagca tcacggtggt ctgtgcccag
      661 ggcttgcagg ccaaggacaa gacaggatcc agtgaccctt atgtcaccgt ccaggttggg
      721 aagaccaaga aaaggacaaa aaccatctac gggaacctta acccagtgtg ggaagagaat
      781 ttccactttg aatgtcacaa ctcctctgac cggatcaagg tgcgtgtgtg ggatgaagac
      841 gatgacatca aatcccgtgt gaagcaaagg tttaagaggg agtctgatga cttcctaggg
      901 cagacaatca tcgaggtgcg gacgcttagc ggcgagatgg atgtgtggta caatctggac
      961 aagagaacag acaagtctgc tgtgtcgggc gccattcggc ttcacatcag tgtggagatc
     1021 aaaggggagg agaaggtggc accctaccat gtccagtaca cctgtctgca tgagaacctg
     1081 ttccattttg tgacggatgt acagaacaat ggcgtggtga agattcccga tgccaaaggt
     1141 gacgacgcct ggaaggttta ctatgatgag actgcccagg agatcgtgga tgagtttgcc
     1201 atgcgctatg gcgttgaatc catctaccaa gccatgaccc actttgcctg cctctcctcc
     1261 aagtacatgt gccctggggt acccgctgtc atgagcaccc tgcttgccaa catcaacgcc
     1321 tactacgcac acaccaccgc ctccaccaac gtgtctgcct ccgaccgctt cgctgcctcc
     1381 aatttcggga aagagcgctt tgtgaaactt ctggaccagc tgcacaactc cctgcggatt
     1441 gacctgtcca tgtaccggaa caacttcccg gccagcagcc ccgagcggct gcaggatctc
     1501 aagtccacag tggacctgct taccagcatc accttcttcc ggatgaaggt tcaggaactg
     1561 cagagtccgc cacgtgccag tcaggtggtg aaggactgcg tgaaggcgtg cctcaactcc
     1621 acctacgagt acatcttcaa caactgtcat gagctctatg gccgggagta ccagacagac
     1681 ccggccaaga agggggaggt ccccccggag gagcaaggcc ctagcatcaa gaacctggat
     1741 ttctggtcca agctcatcac cctcatcgtg tctatcattg aggaggataa gaactcctac
     1801 acaccctgcc tcaatcagtt tccccaggag ctgaatgtgg ggaagatcag tgctgaggtg
     1861 atgtggagcc tgtttgccca ggacatgaag tacgccatgg aggaacacga caagcaccgg
     1921 ctgtgtaaga gcgcagacta catgaacctg catttcaagg tcaagtggtt gtacaacgag
     1981 tacgtggctg aactgcccac cttcaaggac cgcgtgccgg agtaccctgc gtggtttgag
     2041 cccttcgtca tccagtggtt agatgagaat gaggaggtgt cccgggactt cctgcatggt
     2101 gcactcgagc gggacaagaa ggatgggttc cagcagacat cggagcacgc cctgttctct
     2161 tgctcggtag tggacgtctt ctcccagctc aaccagagct ttgagatcat caagaagctg
     2221 gagtgtcctg acccccagat tgtgggccac tacatgcggc gctttgccaa gaccattagc
     2281 aatgtgcttc tccagtacgc cgacatcgtc tccaaggact tcgcttccta ctgctccaag
     2341 gagaaggaga aagtgccctg catcctcatg aacaacacac agcagctgcg ggtgcagctg
     2401 gagaagatgt tcgaggcgat gggtgggaag gagctggacg ccgaggccag tggaaccctg
     2461 aaggagctgc aggtgaaact caacaatgtc ctggatgaac tcagccacgt gtttgccacc
     2521 agcttccagc cacacatcga ggagtgtgtc agacaaatgg gtgacatcct aagccaagtg
     2581 aagggcacgg gcaacgtgcc cgccagtgcc tgcagcagcg tggcacagga cgcagacaac
     2641 gtgctacagc ccatcatgga tcttctggac agcaacctca ccctgtttgc caaaatctgt
     2701 gagaagacgg ttctgaagcg ggtgctgaag gagctgtgga agctggtgat gaacaccatg
     2761 gagaggacca ttgtcctgcc gccatccaaa ctgaaggatc acatggtgcg agaagaagcc
     2821 aagagcttga ccccgaagca gtgtgccgtg gttgaactgg ccctggacac catcaagcaa
     2881 tacttccacg cggggggcgt gggcctcaag aagacctttc tcgagaaaag cccggacctt
     2941 cagtccctgc gctacgccct gtcgctctac acacaggcca ccgacctgct catcaaaacc
     3001 ttcgtgcaga cgcagtcagc gcagggctcg ggtgtggaag accctgtagg tgaagtatcc
     3061 gtccacgtgg agctgttcac gcatccggga actggggaac agaaggtcac agtgaaggtg
     3121 gtggccgcca acgacctcaa gtggcagact tctggcatct tcgaaccgtt cattgaggtc
     3181 aacatcgttg gacctcagct cagcgacaag aaacgaaagt tcgccaccaa atcgaaaaac
     3241 aacagctggg cgcccaaata taacgagagc ttccagttct ccctgagcgc cgacgcggga
     3301 cccgagtgct acgagttgca ggtgtgcgtg aaggactact gcgaagcgcg cgaggaccgc
     3361 acggtggggc tggcggtgct gcagctgcgg gagctggctc agcgcgggag cgccgcgtgc
     3421 tggctgccgc tcggccgccg catccacatg gacgacacgg ggctcacagt gctgcgtatc
     3481 ctgtcgcagc gcagcaatga cgaggtggcc aaggagttcg tcaagctcaa gtccgacacg
     3541 cgctcggccg aggagggcgg tgccgcgcct gcgccctaaa agcttgcggc cgcactcgag
     3601 caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg
     3661 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     3721 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
     3781 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     3841 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     3901 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     3961 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     4021 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     4081 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     4141 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     4201 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     4261 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga
     4321 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat
     4381 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga
     4441 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta
     4501 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat
     4561 ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat
     4621 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct
     4681 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca
     4741 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt
     4801 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag
     4861 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc
     4921 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact
     4981 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat
     5041 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag
     5101 agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag
     5161 cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     5221 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     5281 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     5341 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     5401 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     5461 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     5521 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     5581 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     5641 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     5701 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     5761 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     5821 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     5881 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     5941 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     6001 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     6061 ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     6121 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     6181 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     6241 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     6301 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg
     6361 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa
     6421 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg
     6481 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt
     6541 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg
     6601 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga
     6661 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt
     6721 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca
     6781 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat
     6841 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga
     6901 caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc
     6961 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc
     7021 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa
     7081 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat
     7141 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc
     7201 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
     7261 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     7321 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     7381 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     7441 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     7501 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     7561 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     7621 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     7681 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     7741 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     7801 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     7861 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     7921 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     7981 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     8041 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     8101 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     8161 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     8221 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     8281 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     8341 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     8401 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     8461 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
     8521 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     8581 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
     8641 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
     8701 ccggcgtaga ggatcgagat ctcgatcccg cgaaat
//
