LOCUS       sequence-486413-        6845 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 6845)
  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 6845)
  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: GST-Thrombin-Munc13-1 MUN domain (aa859-1516
            d...
FEATURES             Location/Qualifiers
     source          1..6845
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..22,23..29)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..53
                     /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             76..729
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     687..709
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             736..753
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(2713..2735)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2880..2898)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2966..3070
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3071..3139,3140..3931)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3289..3308)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4102..4690
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4591..4610
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4844..4861
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4934..5011
                     /gene="lacI"
                     /label=lacI promoter
     CDS             5012..6094
                     /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     complement(5031..5050)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(6143..6148,6149..6166,6167..6173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6181..6197
                     /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)."
     primer_bind     6205..6221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6205..6221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             6217..6744
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(6237..6254)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6237..6253)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6246..6268)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag tattcatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa
      121 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc
      181 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt
      241 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata
      301 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt
      361 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt
      421 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat
      481 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg
      541 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa
      601 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc
      661 agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat
      721 cctccaaaat cggatctggt tccgcgtgga tccatgggtg acgacgcctg gaaggtttac
      781 tatgatgaga ctgcccagga gatcgtggat gagtttgcca tgcgctatgg cgttgaatcc
      841 atctaccaag ccatgaccca ctttgcctgc ctctcctcca agtacatgtg ccctggggta
      901 cccgctgtca tgagcaccct gcttgccaac atcaacgcct actacgcaca caccaccgcc
      961 tccaccaacg tgtctgcctc cgaccgcttc gctgcctcca atttcgggaa agagcgcttt
     1021 gtgaaacttc tggaccagct gcacaactcc ctgcggattg acctgtccat gtaccggaac
     1081 aacttcccgg ccagcagccc cgagcggctg caggatctca agtccacagt ggacctgctt
     1141 accagcatca ccttcttccg gatgaaggtt caggaactgc agagtccgcc acgtgccagt
     1201 caggtggtga aggactgcgt gaaggcgtgc ctcaactcca cctacgagta catcttcaac
     1261 aactgtcatg agctctatgg ccgggagtac cagacagacc cggccaagaa gggggaggtc
     1321 cccccggagg agcaaggccc tagcatcaag aacctggatt tctggtccaa gctcatcacc
     1381 ctcatcgtgt ctatcattga ggaggataag aactcctaca caccctgcct caatcagttt
     1441 ccccaggagc tgaatgtggg gaagatcagt gctgaggtga tgtggagcct gtttgcccag
     1501 gacatgaagt acgccatgga ggaacacgac aagcaccggc tgtgtaagag cgcagactac
     1561 atgaacctgc atttcaaggt caagtggttg tacaacgagt acgtggctga actgcccacc
     1621 ttcaaggacc gcgtgccgga gtaccctgcg tggtttgagc ccttcgtcat ccagtggtta
     1681 gatgagaatg aggaggtgtc ccgggacttc ctgcatggtg cactcgagcg ggacaagaag
     1741 gatgggttcc agcagacatc ggagcacgcc ctgttctctt gctcggtagt ggacgtcttc
     1801 tcccagctca accagagctt tgagatcatc aagaagctgg agtgtcctga cccccagatt
     1861 gtgggccact acatgcggcg ctttgccaag accattagca atgtgcttct ccagtacgcc
     1921 gacatcgtct ccaaggactt cgcttcctac tgctccaagg agaaggagaa agtgccctgc
     1981 atcctcatga acaacacaca gcagctgcgg gtgcagctgg agaagatgtt cgaggcgatg
     2041 ggtgggaagg agctggacgc cgaggccagt ggaaccctga aggagctgca ggtgaaactc
     2101 aacaatgtcc tggatgaact cagccacgtg tttgccacca gcttccagcc acacatcgag
     2161 gagtgtgtca gacaaatggg tgacatccta agccaagtga agggcacggg caacgtgccc
     2221 gccagtgcct gcagcagcgt ggcacaggac gcagacaacg tgctacagcc catcatggat
     2281 cttctggaca gcaacctcac cctgtttgcc aaaatctgtg agaagacggt tctgaagcgg
     2341 gtgctgaagg agctgtggaa gctggtgatg aacaccatgg agaggaccat tgtcctgccg
     2401 ccagaattct ccaaactgaa ggatcacatg gtgcgagaag aagccaagag cttgaccccg
     2461 aagcagtgtg ccgtggttga actggccctg gacaccatca agcaatactt ccacgcgggg
     2521 ggcgtgggcc tcaagaagac ctttctcgag aaaagcccgg accttcagtc cctgcgctac
     2581 gccctgtcgc tctacacaca gtaaaccgac ctgctcatca aaaccttcgt gcagacgcag
     2641 tcagcgtaaa agcttaattc atcgtgactg actgacgatc tgcctcgcgc gtttcggtga
     2701 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
     2761 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
     2821 cgcagccatg acccagtcac gtagcgatag cggagtgtat aattcttgaa gacgaaaggg
     2881 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     2941 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     3001 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     3061 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     3121 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     3181 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     3241 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     3301 ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     3361 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     3421 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     3481 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     3541 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     3601 caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     3661 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     3721 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     3781 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     3841 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     3901 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     3961 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     4021 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     4081 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     4141 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     4201 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     4261 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     4321 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     4381 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     4441 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     4501 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     4561 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     4621 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     4681 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4741 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     4801 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     4861 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     4921 ccgcataaat tccgacacca tcgaatggcg caaaaccttt cgcggtatgg catgatagcg
     4981 cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc
     5041 agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt
     5101 ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa
     5161 ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag
     5221 tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact
     5281 gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc
     5341 ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga
     5401 ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt
     5461 ctctgaccag acacccatca acagtattat tttctcccat gaagacggta cgcgactggg
     5521 cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag
     5581 ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat
     5641 tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat
     5701 gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc
     5761 gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt
     5821 agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa
     5881 acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg
     5941 ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct
     6001 ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     6061 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc
     6121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
     6181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac ggattcactg
     6241 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     6301 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     6361 tcccaacagt tgcgcagcct gaatggcgaa tggcgctttg cctggtttcc ggcaccagaa
     6421 gcggtgccgg aaagctggct ggagtgcgat cttcctgagg ccgatactgt cgtcgtcccc
     6481 tcaaactggc agatgcacgg ttacgatgcg cccatctaca ccaacgtgac ctatcccatt
     6541 acggtcaatc cgccgtttgt tcccacggag aatccgacgg gttgttactc gctcacattt
     6601 aatgttgatg aaagctggct acaggaaggc cagacgcgaa ttatttttga tggcgttgga
     6661 attagcttat cgactgcacg gtgcaccaat gcttctggcg tcaggcagcc atcggaagct
     6721 gtggtatggc tgtgcaggtc gtaaatcact gcataattcg tgtcgctcaa ggcgcactcc
     6781 cgttctggat aatgtttttt gcgccgacat cataacggtt ctggcaaata ttctgaaatg
     6841 agctg
//
