LOCUS       sequence-490982-        8739 bp    DNA     circular SYN 29-OCT-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 Oct 29, 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"
     protein_bind    19..43
                     /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             58..80
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             99..116
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             126..143
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3603..3620
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3673..3691)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3687..3734
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3771..4226
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3858..3877)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4068..4089
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4318..5133)
                     /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(4318..5133)
                     /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     5041..5060
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5125..5144
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5255..5843
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5744..5763
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5997..6014
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6029..6168
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6094..6113)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6254..6276
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6270..6461)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(7270..8352)
                     /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     8314..8333
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8353..8430)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     8636..8655
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(8739,1..19)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(8739,1..18)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt
       61 gtttaacttt aagaaggaga tataccatgg gcagcagcca tcatcatcat catcacagca
      121 gcggcctggt gccgcgcggc agccatatgg cgggcatcac ctcggccttg gcctccagca
      181 cgttgaacaa tgaagagttg aaaaaccacg tttacaagaa gaccctgcaa gccttaatct
      241 accccatctc ctgcaccacg ccgcacaact tcgaggtgtg gacggccacc actcccacct
      301 actgctacga gtgcgagggg ctgctgtggg gcatcgcgcg gcagggcatg cgctgcaccg
      361 agtgcggcgt taagtgccac gagaagtgcc aagacctgct caacgcggac tgcctgcagc
      421 gggcggctga gaagagttct aagcatggcg ctgaagaccg cacgcagaac atcatcatgg
      481 tgctgaagga ccgcatgaag atccgcgagc gcaacaagcc tgagatcttc gagctgatcc
      541 aggaggtctt cgcggtcacc aagagcgcac acacacagca gatgaaggcc gtcaagcaga
      601 gtgtgctgga tggcacatcc aagtggtctg ccaaaattag catcacggtg gtctgtgccc
      661 agggcttgca ggccaaggac aagacaggat ccagtgaccc ttatgtcacc gtccaggttg
      721 ggaagaccaa gaaaaggaca aaaaccatct acgggaacct taacccagtg tgggaagaga
      781 atttccactt tgaatgtcac aactcctctg accggatcaa ggtgcgtgtg tgggatgaag
      841 acgatgacat caaatcccgt gtgaagcaaa ggtttaagag ggagtctgat gacttcctag
      901 ggcagacaat catcgaggtg cggacgctta gcggcgagat ggatgtgtgg tacaatctgg
      961 acaagagaac agacaagtct gctgtgtcgg gcgccattcg gcttcacatc agtgtggaga
     1021 tcaaagggga ggagaaggtg gcaccctacc atgtccagta cacctgtctg catgagaacc
     1081 tgttccattt tgtgacggat gtacagaaca atggcgtggt gaagattccc gatgccaaag
     1141 gtgacgacgc ctggaaggtt tactatgatg agactgccca ggagatcgtg gatgagtttg
     1201 ccatgcgcta tggcgttgaa tccatctacc aagccatgac ccactttgcc tgcctctcct
     1261 ccaagtacat gtgccctggg gtacccgctg tcatgagcac cctgcttgcc aacatcaacg
     1321 cctactacgc acacaccacc gcctccacca acgtgtctgc ctccgaccgc ttcgctgcct
     1381 ccaatttcgg gaaagagcgc tttgtgaaac ttctggacca gctgcacaac tccctgcgga
     1441 ttgacctgtc catgtaccgg aacaacttcc cggccagcag ccccgagcgg ctgcaggatc
     1501 tcaagtccac agtggacctg cttaccagca tcaccttctt ccggatgaag gttcaggaac
     1561 tgcagagtcc gccacgtgcc agtcaggtgg tgaaggactg cgtgaaggcg tgcctcaact
     1621 ccacctacga gtacatcttc aacaactgtc atgagctcta tggccgggag taccagacag
     1681 acccggccaa gaagggggag gtccccccgg aggagcaagg ccctagcatc aagaacctgg
     1741 atttctggtc caagctcatc accctcatcg tgtctatcat tgaggaggat aagaactcct
     1801 acacaccctg cctcaatcag tttccccagg agctgaatgt ggggaagatc agtgctgagg
     1861 tgatgtggag cctgtttgcc caggacatga agtacgccat ggaggaacac gacaagcacc
     1921 ggctgtgtaa gagcgcagac tacatgaacc tgcatttcaa ggtcaagtgg ttgtacaacg
     1981 agtacgtggc tgaactgccc accttcaagg accgcgtgcc ggagtaccct gcgtggtttg
     2041 agcccttcgt catccagtgg ttagatgaga atgaggaggt gtcccgggac ttcctgcatg
     2101 gtgcactcga gcgggacaag aaggatgggt tccagcagac atcggagcac gccctgttct
     2161 cttgctcggt agtggacgtc ttctcccagc tcaaccagag ctttgagatc atcaagaagc
     2221 tggagtgtcc tgacccccag attgtgggcc actacatgcg gcgctttgcc aagaccatta
     2281 gcaatgtgct tctccagtac gccgacatcg tctccaagga cttcgcttcc tactgctcca
     2341 aggagaagga gaaagtgccc tgcatcctca tgaacaacac acagcagctg cgggtgcagc
     2401 tggagaagat gttcgaggcg atgggtggga aggagctgga cgccgaggcc agtggaaccc
     2461 tgaaggagct gcaggtgaaa ctcaacaatg tcctggatga actcagccac gtgtttgcca
     2521 ccagcttcca gccacacatc gaggagtgtg tcagacaaat gggtgacatc ctaagccaag
     2581 tgaagggcac gggcaacgtg cccgccagtg cctgcagcag cgtggcacag gacgcagaca
     2641 acgtgctaca gcccatcatg gatcttctgg acagcaacct caccctgttt gccaaaatct
     2701 gtgagaagac ggttctgaag cgggtgctga aggagctgtg gaagctggtg atgaacacca
     2761 tggagaggac cattgtcctg ccgccatcca aactgaagga tcacatggtg cgagaagaag
     2821 ccaagagctt gaccccgaag cagtgtgccg tggttgaact ggccctggac accatcaagc
     2881 aatacttcca cgcggggggc gtgggcctca agaagacctt tctcgagaaa agcccggacc
     2941 ttcagtccct gcgctacgcc ctgtcgctct acacacaggc caccgacctg ctcatcaaaa
     3001 ccttcgtgca gacgcagtca gcgcagggct cgggtgtgga agaccctgta ggtgaagtat
     3061 ccgtccacgt ggagctgttc acgcatccgg gaactgggga acagaaggtc acagtgaagg
     3121 tggtggccgc caacgacctc aagtggcaga cttctggcat cttccgtccg ttcattgagg
     3181 tcaacatcgt tggacctcag ctcagcgaca agaaacgaaa gttcgccacc aaatcgaaaa
     3241 acaacagctg ggcgcccaaa tataacgaga gcttccagtt ctccctgagc gccgacgcgg
     3301 gacccgagtg ctacgagttg caggtgtgcg tgaaggacta ctgcttcgcg cgcgaggacc
     3361 gcacggtggg gctggcggtg ctgcagctgc gggagctggc tcagcgcggg agcgccgcgt
     3421 gctggctgcc gctcggccgc cgcatccaca tggacgacac ggggctcaca gtgctgcgta
     3481 tcctgtcgca gcgcagcaat gacgaggtgg ccaaggagtt cgtcaagctc aagtccgaca
     3541 cgcgctcggc cgaggagggc ggtgccgcgc ctgcgcccta aaagcttgcg gccgcactcg
     3601 agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt
     3661 tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
     3721 tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg
     3781 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     3841 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     3901 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     3961 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     4021 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     4081 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     4141 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     4201 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
     4261 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta
     4321 gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc
     4381 atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag
     4441 gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat
     4501 taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga
     4561 atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc
     4621 attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc
     4681 ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg
     4741 caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc
     4801 ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc
     4861 aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag
     4921 tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa
     4981 ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt
     5041 atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta atcgcggcct
     5101 agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta
     5161 agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     5221 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     5281 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     5341 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     5401 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     5461 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     5521 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     5581 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     5641 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     5701 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     5761 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     5821 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     5881 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     5941 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     6001 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     6061 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     6121 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     6181 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     6241 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     6301 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     6361 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     6421 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     6481 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     6541 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     6601 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     6661 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     6721 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     6781 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     6841 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     6901 gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc
     6961 tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt
     7021 ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg
     7081 aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc
     7141 ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     7201 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt
     7261 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     7321 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     7381 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     7441 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     7501 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc
     7561 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     7621 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
     7681 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
     7741 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     7801 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     7861 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     7921 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     7981 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     8041 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     8101 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     8161 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     8221 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     8281 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     8341 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     8401 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact
     8461 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
     8521 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
     8581 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
     8641 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
     8701 gtccggcgta gaggatcgag atctcgatcc cgcgaaatt
//
