LOCUS       sequence-440961-        6483 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses rat MUNC18a in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6483)
  AUTHORS   Leitz J, Wang C, Esquivies L, Peters JJ, Gopal N, Pfuetzner RA, Wang
            AL, Brunger AT
  TITLE     Observing isolated synaptic vesicle association and fusion ex vivo.
  JOURNAL   Nat Protoc. 2024 Jul 2. doi: 10.1038/s41596-024-01014-x.
  PUBMED    38956381
REFERENCE   2  (bases 1 to 6483)
  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"; journalName: "Nat Protoc.
            2024 Jul 2. doi: 10.1038/s41596-024-01014-x."
COMMENT     Sequence Label: MUNC18a
FEATURES             Location/Qualifiers
     source          1..6483
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    230..246
                     /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     235..257
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             279..296
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             318..338
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(2205..2222)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2375..2461
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2553..2580
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2599..2690
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2691..2759,2760..3551)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2909..2928)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3722..4310
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4211..4230
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4464..4481
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4558..4986)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4668..4689)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4880..4899
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(5024..5043)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5184..5206
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        5284..5361
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             5362..6444
                     /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(5381..5400)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gtttgacagc ttatcatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc
       61 ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc
      121 gcactcccgt tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc
      181 tgaaatgagc tgttgacaat taatcatccg gtccgtataa tctgtggaat tgtgagcgga
      241 taacaatttc acacaggaaa cagaccatgt cgtactacca tcaccatcac catcacgatt
      301 acgatatccc aacgaccgaa aacctgtatt ttcagggcgc catggatatg gcccccattg
      361 gcctcaaggc ggtggtcgga gagaagatca tgcatgatgt tatcaagaag gtgaagaaga
      421 agggcgagtg gaaggtgctg gtggtggacc agttaagcat gaggatgctg tcttcctgct
      481 gcaagatgac agacatcatg accgagggga tcacaattgt ggaggatatc aacaagcgcc
      541 gagagccgct ccccagccta gaggccgtgt acctcatcac cccgtctgag aagtctgtcc
      601 actctctcat cagtgatttt aaggacccgc cgactgctaa atatcgggcc gcacatgtgt
      661 tcttcacaga ctcatgtcca gatgccctgt ttaacgagct ggtaaaatct cgagcagcca
      721 aagtcatcaa gacgctgacg gaaatcaaca ttgcgtttct cccctatgag tcccaggtgt
      781 attccctgga ctccgctgac tctttccaaa gtttctacag ccctcacaag gcacagatga
      841 agaatcccat actggaacgc ctggcagagc aaatcgccac cctgtgtgcc accctgaagg
      901 agtacccggc tgtgcggtat cggggggagt acaaggacaa tgccttgctg gctcagctga
      961 tccaggacaa gctggacgct tataaagctg atgacccgac aatgggggag ggtcccgaca
     1021 aggcacggtc ccagctcctg atcctggatc gtggctttga ccccagctcc cctgtgcttc
     1081 atgaactgac gtttcaggct atgagttacg acctgctgcc tattgaaaat gatgtttaca
     1141 agtatgagac cagcggcatt ggagaggcga gggtgaagga ggtgctcctg gatgaggacg
     1201 atgacctgtg gattgcactg cgacacaagc acatcgcaga ggtgtcccag gaagtgaccc
     1261 ggtctctgaa ggacttttcc tctagcaaga ggatgaatac tggagagaag accaccatgc
     1321 gggacctgtc ccagatgctg aagaaaatgc cccagtacca gaaggagctc agcaagtatt
     1381 cgactcacct gcaccttgct gaagactgta tgaagcatta ccaaggcacc gtagacaaac
     1441 tctgccgcgt ggagcaggac ctggcgatgg gcacagatgc tgagggggaa aaaatcaagg
     1501 accccatgag agccattgtc cccatcctgc tggatgcaaa tgtcagcact tatgacaaaa
     1561 ttcgcatcat ccttctctac atcttcctga agaacggtat cactgaagaa aacctaaaca
     1621 aactcatcca gcacgcccag atccccccag aagacagtga gatcatcacc aacatggctc
     1681 acctcggggt gcccatcgtc acggattcca cactacgccg cagaagcaag ccggagcgga
     1741 aggagcgcat cagtgagcag acctaccagc tctcacgatg gaccccgatc attaaagaca
     1801 ttatggagga caccattgaa gacaaactgg atacaaagca ctacccatac atctctaccc
     1861 gctcttccgc atccttcagc accactgctg tgagtgcccg ctatggacat tggcacaaga
     1921 ataaggcccc cggggagtac cgcagcggcc cccgcctcat tattttcatc cttgggggcg
     1981 tgagcctgaa tgagatgcgc tgtgcctacg aagtgacgca ggccaacggc aagtgggaag
     2041 tgctcatagg atccacgcac attctcaccc cacagaaact gctggacacg ctgaagaagc
     2101 tgaataaaac agacgaagaa ataagcagtg gtagtggttg actgcagtct cgaggcatgc
     2161 ggtaccaagc ttggctgttt tggcggatga gagaagattt tcagcctgat acagattaaa
     2221 tcagaacgca gaagcggtct gataaaacag aatttgcctg gcggcagtag cgcggtggtc
     2281 ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg
     2341 tctccccatg cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa
     2401 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa
     2461 tccgccggga gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg
     2521 cccgccataa actgccaggc atcaaattaa gcagaaggcc atcctgacgg atggcctttt
     2581 tgcgtttcta caaactcttt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     2641 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     2701 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     2761 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     2821 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     2881 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg
     2941 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     3001 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     3061 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     3121 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     3181 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     3241 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     3301 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     3361 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     3421 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     3481 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     3541 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     3601 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     3661 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     3721 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     3781 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     3841 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     3901 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     3961 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     4021 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     4081 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     4141 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     4201 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     4261 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     4321 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     4381 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     4441 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     4501 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcataatt ttgttaaaat
     4561 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     4621 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     4681 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     4741 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     4801 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     4861 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     4921 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     4981 gcgcgtccca ttcgccattc aggctgctat ggtgcactct cagtacaatc tgctctgatg
     5041 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     5101 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     5161 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     5221 accgaaacgc gcgaggcagc agatcaattc gcgcgcgaag gcgaagcggc atgcatttac
     5281 gttgacacca tcgaatggtg caaaaccttt cgcggtatgg catgatagcg cccggaagag
     5341 agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc
     5401 ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa
     5461 acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca
     5521 caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg
     5581 cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc
     5641 gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat
     5701 cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc
     5761 attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag
     5821 acacccatca acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat
     5881 ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg
     5941 gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata
     6001 gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg
     6061 aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca
     6121 atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt agtgggatac
     6181 gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa acaggatttt
     6241 cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg
     6301 aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggcacccaat
     6361 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     6421 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc gcgaattgat
     6481 ctg
//
