LOCUS       sequence-445909-        7365 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the MUN domain from MUNC13-1 in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7365)
  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 7365)
  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: MUNC13-1 MUN
FEATURES             Location/Qualifiers
     source          1..7365
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             33..50
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             72..92
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(2016..2034)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2030..2077
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2114..2569
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2201..2220)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2411..2432
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2595..2699
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2700..2768,2769..3560)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2918..2937)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     primer_bind     4301..4320
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4554..4571
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4586..4725
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4651..4670)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4811..4833
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4827..5018)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5827..6909)
                     /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     6871..6890
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6910..6987)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7193..7212
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     7296..7315
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7296..7314
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7315..7339
                     /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             join(7354..7365,1..11)
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 taagaaggag atatacatat atgtcgtact accatcacca tcaccatcac gattacgata
       61 tcccaactag tgaaaacctg tattttcagg gcgctagcat gggtgacgac gcctggaagg
      121 tttactatga tgagactgcc caggagatcg tggatgagtt tgccatgcgc tatggcgttg
      181 aatccatcta ccaagccatg acccactttg cctgcctctc ctccaagtac atgtgccctg
      241 gggtacccgc tgtcatgagc accctgcttg ccaacatcaa cgcctactac gcacacacca
      301 ccgcctccac caacgtgtct gcctccgacc gcttcgctgc ctccaatttc gggaaagagc
      361 gctttgtgaa acttctggac cagctgcaca actccctgcg gattgacctg tccatgtacc
      421 ggaacaactt cccggccagc agccccgagc ggctgcagga tctcaagtcc acagtggacc
      481 tgcttaccag catcaccttc ttccggatga aggttcagga actgcagagt ccgccacgtg
      541 ccagtcaggt ggtgaaggac tgcgtgaagg cgtgcctcaa ctccacctac gagtacatct
      601 tcaacaactg tcatgagctc tatggccggg agtaccagac agacccggcc aagaaggggg
      661 aggtcccccc ggaggagcaa ggccctagca tcaagaacct ggatttctgg tccaagctca
      721 tcaccctcat cgtgtctatc attgaggagg ataagaactc ctacacaccc tgcctcaatc
      781 agtttcccca ggagctgaat gtggggaaga tcagtgctga ggtgatgtgg agcctgtttg
      841 cccaggacat gaagtacgcc atggaggaac acgacaagca ccggctgtgt aagagcgcag
      901 actacatgaa cctgcatttc aaggtcaagt ggttgtacaa cgagtacgtg gctgaactgc
      961 ccaccttcaa ggaccgcgtg ccggagtacc ctgcgtggtt tgagcccttc gtcatccagt
     1021 ggttagatga gaatgaggag gtgtcccggg acttcctgca tggtgcactc gagcgggaca
     1081 agaaggatgg gttccagcag acatcggagc acgccctgtt ctcttgctcg gtagtggacg
     1141 tcttctccca gctcaaccag agctttgaga tcatcaagaa gctggagtgt cctgaccccc
     1201 agattgtggg ccactacatg cggcgctttg ccaagaccat tagcaatgtg cttctccagt
     1261 acgccgacat cgtctccaag gacttcgctt cctactgctc caaggagaag gagaaagtgc
     1321 cctgcatcct catgaacaac acacagcagc tgcgggtgca gctggagaag atgttcgagg
     1381 cgatgggtgg gaaggagctg gacgccgagg ccagtggaac cctgaaggag ctgcaggtga
     1441 aactcaacaa tgtcctggat gaactcagcc acgtgtttgc caccagcttc cagccacaca
     1501 tcgaggagtg tgtcagacaa atgggtgaca tcctaagcca agtgaagggc acgggcaacg
     1561 tgcccgccag tgcctgcagc agcgtggcac aggacgcaga caacgtgcta cagcccatca
     1621 tggatcttct ggacagcaac ctcaccctgt ttgccaaaat ctgtgagaag acggttctga
     1681 agcgggtgct gaaggagctg tggaagctgg tgatgaacac catggagagg accattgtcc
     1741 tgccgccatc caaactgaag gatcacatgg tgcgagaaga agccaagagc ttgaccccga
     1801 agcagtgtgc cgtggttgaa ctggccctgg acaccatcaa gcaatacttc cacgcggggg
     1861 gcgtgggcct caagaagacc tttctcgaga aaagcccgga ccttcagtcc ctgcgctacg
     1921 ccctgtcgct ctacacacag gccaccgacc tgctcatcaa aaccttcgtg cagacgcagt
     1981 cagcgtaaga attctcgagc tcccgggatc cgcggccgct gagcaataac tagcataacc
     2041 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     2101 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     2161 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     2221 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     2281 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     2341 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     2401 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     2461 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     2521 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     2581 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     2641 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     2701 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     2761 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     2821 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     2881 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     2941 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     3001 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     3061 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     3121 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     3181 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     3241 ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     3301 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     3361 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     3421 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     3481 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     3541 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     3601 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     3661 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     3721 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     3781 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     3841 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     3901 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     3961 cagtggatcg acggagcgat gccgagccgt gctgaaacgg taaaaggtta cgtgcaggcc
     4021 atgcaccaga gctggttaca ggatgtgctg ctgccagtgg cgataagtcg tgtcttaccg
     4081 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     4141 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     4201 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     4261 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     4321 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     4381 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     4441 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     4501 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     4561 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     4621 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     4681 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     4741 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     4801 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     4861 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     4921 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     4981 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     5041 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     5101 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     5161 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     5221 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     5281 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     5341 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     5401 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     5461 aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg
     5521 ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg
     5581 aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa
     5641 atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata
     5701 agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     5761 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     5821 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     5881 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     5941 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     6001 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     6061 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac
     6121 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     6181 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     6241 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     6301 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     6361 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     6421 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     6481 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     6541 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     6601 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     6661 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     6721 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     6781 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     6841 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     6901 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     6961 aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct
     7021 gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg
     7081 gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca
     7141 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt
     7201 cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc
     7261 cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta taggggaatt
     7321 gtgagcggat aacaattccc ctctagaaat aattttgttt aactt
//
