LOCUS       sequence-435306-        8774 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Insect cell expression of a rat MUNC13-1 fragment containing domains
            C1C2BMUNC2C.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8774)
  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 8774)
  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: C1C2CMUNC2C
FEATURES             Location/Qualifiers
     source          1..8774
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(79..98)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     288..309
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        472..576
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(577..645,646..1437)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(795..814)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1608..2196
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2097..2116
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2350..2367
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  2501..2725
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(2792..3325)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(3495..3500,3501..3517,3518..3523))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     promoter        3875..3966
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     3891..3908
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     primer_bind     4620..4642
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             4648..4665
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             4666..4686
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     polyA_signal    8254..8388
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8291..8310)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8345..8364
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  8417..8582
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      join(8766..8774,1..446)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
       61 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
      121 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
      181 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
      241 gatagacggt ttttcgccct ttgacgttgg agtccacgtt cttaatagtg gactcttgtt
      301 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
      361 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
      421 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
      481 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
      541 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
      601 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
      661 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
      721 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
      781 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
      841 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
      901 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
      961 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     1021 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     1081 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     1141 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     1201 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     1261 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     1321 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     1381 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     1441 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     1501 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     1561 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     1621 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     1681 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     1741 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     1801 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     1861 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     1921 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     1981 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     2041 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     2101 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     2161 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     2221 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     2281 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     2341 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     2401 cttacgcatc tgtgcggtat ttcacaccgc atagaccagc cgcgtaacct ggcaaaatcg
     2461 gttacggttg agtaataaat ggatgccctg cgtaagcggg tgtgggcgga caataaagtc
     2521 ttaaactgaa caaaatagat ctaaactatg acaataaagt cttaaactag acagaatagt
     2581 tgtaaactga aatcagtcca gttatgctgt gaaaaagcat actggacttt tgttatggct
     2641 aaagcaaact cttcattttc tgaagtgcaa attgcccgtc gtattaaaga ggggcgtggc
     2701 caagggcatg gtaaagacta tattcgcggc gttgtgacaa tttaccgaac aactccgcgg
     2761 ccgggaagcc gatctcggct tgaacgaatt gttaggtggc ggtacttggg tcgatatcaa
     2821 agtgcatcac ttcttcccgt atgcccaact ttgtatagag agccactgcg ggatcgtcac
     2881 cgtaatctgc ttgcacgtag atcacataag caccaagcgc gttggcctca tgcttgagga
     2941 gattgatgag cgcggtggca atgccctgcc tccggtgctc gccggagact gcgagatcat
     3001 agatatagat ctcactacgc ggctgctcaa acttgggcag aacgtaagcc gcgagagcgc
     3061 caacaaccgc ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta cggagcaagt
     3121 tcccgaggta atcggagtcc ggctgatgtt gggagtaggt ggctacgtct ccgaactcac
     3181 gaccgaaaag atcaagagca gcccgcatgg atttgacttg gtcagggccg agcctacatg
     3241 tgcgaatgat gcccatactt gagccaccta actttgtttt agggcgactg ccctgctgcg
     3301 taacatcgtt gctgctccat aacatcaaac atcgacccac ggcgtaacgc gcttgctgct
     3361 tggatgcccg aggcatagac tgtacaaaaa aacagtcata acaagccatg aaaaccgcca
     3421 ctgcgccgtt accaccgctg cgttcggtca aggttctgga ccagttgcgt gagcgcatac
     3481 gctacttgca ttacagttta cgaaccgaac aggcttatgt caactgggtt cgtgccttca
     3541 tccgtttcca cggtgtgcgt cacccggcaa ccttgggcag cagcgaagtc gaggcatttc
     3601 tgtcctggct ggcgaacgag cgcaaggttt cggtctccac gcatcgtcag gcattggcgg
     3661 ccttgctgtt cttctacggc aaggtgctgt gcacggatct gccctggctt caggagatcg
     3721 gaagacctcg gccgtcgcgg cgcttgccgg tggtgctgac cccggatgaa gtggttcgca
     3781 tcctcggttt tctggaaggc gagcatcgtt tgttcgccca ggactctagc tatagttcta
     3841 gtggttggct acgtatactc cggaatatta atagatcatg gagataatta aaatgataac
     3901 catctcgcaa ataaataagt attttactgt tttcgtaaca gttttgtaat aaaaaaacct
     3961 ataaatattc cggattattc ataccgtccc accatcgggc gcggatccat gtcccctata
     4021 ctaggttatt ggaaaattaa gggccttgtg caacccactc gacttctttt ggaatatctt
     4081 gaagaaaaat atgaagagca tttgtatgag cgcgatgaag gtgataaatg gcgaaacaaa
     4141 aagtttgaat tgggtttgga gtttcccaat cttccttatt atattgatgg tgatgttaaa
     4201 ttaacacagt ctatggccat catacgttat atagctgaca agcacaacat gttgggtggt
     4261 tgtccaaaag agcgtgcaga gatttcaatg cttgaaggag cggttttgga tattagatac
     4321 ggtgtttcga gaattgcata tagtaaagac tttgaaactc tcaaagttga ttttcttagc
     4381 aagctacctg aaatgctgaa aatgttcaaa gatcgtttat gtcataaaac atatttaaat
     4441 ggtgatcatg taacccatcc tgacttcatg ttgtatgacg ctcttgatgt tgttttatac
     4501 atggacccaa tgtgcctgga tgcgttccca aaattagttt gttttaaaaa acgtattgaa
     4561 gctatcccac aaattgataa gtacttgaaa tccagcaagt atatagcatg gcctttgcag
     4621 ggctggcaag ccacgtttgg tggtggcttg gtccctcgtg gaagcgagaa tctgtacttc
     4681 caaggagaat tcgcgggcat cacctcggcc ttggcctcca gcacgttgaa caatgaagag
     4741 ttgaaaaacc acgtttacaa gaagaccctg caagccttaa tctaccccat ctcctgcacc
     4801 acgccgcaca acttcgaggt gtggacggcc accactccca cctactgcta cgagtgcgag
     4861 gggctgctgt ggggcatcgc gcggcagggc atgcgctgca ccgagtgcgg cgttaagtgc
     4921 cacgagaagt gccaagacct gctcaacgcg gactgcctgc agcgggcggc tgagaagagt
     4981 tctaagcatg gcgctgaaga ccgcacgcag aacatcatca tggtgctgaa ggaccgcatg
     5041 aagatccgcg agcgcaacaa gcctgagatc ttcgagctga tccaggaggt cttcgcggtc
     5101 accaagagcg cacacacaca gcagatgaag gccgtcaagc agagtgtgct ggatggcaca
     5161 tccaagtggt ctgccaaaat tagcatcacg gtggtctgtg cccagggctt gcaggccaag
     5221 gacaagacag gatccagtga cccttatgtc accgtccagg ttgggaagac caagaaaagg
     5281 acaaaaacca tctacgggaa ccttaaccca gtgtgggaag agaatttcca ctttgaatgt
     5341 cacaactcct ctgaccggat caaggtgcgt gtgtgggatg aagacgatga catcaaatcc
     5401 cgtgtgaagc aaaggtttaa gagggagtct gatgacttcc tagggcagac aatcatcgag
     5461 gtgcggacgc ttagcggcga gatggatgtg tggtacaatc tggacaagag aacagacaag
     5521 tctgctgtgt cgggcgccat tcggcttcac atcagtgtgg agatcaaagg ggaggagaag
     5581 gtggcaccct accatgtcca gtacacctgt ctgcatgaga acctgttcca ttttgtgacg
     5641 gatgtacaga acaatggcgt ggtgaagatt cccgatgcca aaggtgacga cgcctggaag
     5701 gtttactatg atgagactgc ccaggagatc gtggatgagt ttgccatgcg ctatggcgtt
     5761 gaatccatct accaagccat gacccacttt gcctgcctct cctccaagta catgtgccct
     5821 ggggtacccg ctgtcatgag caccctgctt gccaacatca acgcctacta cgcacacacc
     5881 accgcctcca ccaacgtgtc tgcctccgac cgcttcgctg cctccaattt cgggaaagag
     5941 cgctttgtga aacttctgga ccagctgcac aactccctgc ggattgacct gtccatgtac
     6001 cggaacaact tcccggccag cagccccgag cggctgcagg atctcaagtc cacagtggac
     6061 ctgcttacca gcatcacctt cttccggatg aaggttcagg aactgcagag tccgccacgt
     6121 gccagtcagg tggtgaagga ctgcgtgaag gcgtgcctca actccaccta cgagtacatc
     6181 ttcaacaact gtcatgagct ctatggccgg gagtaccaga cagacccggc caagaagggg
     6241 gaggtccccc cggaggagca aggccctagc atcaagaacc tggatttctg gtccaagctc
     6301 atcaccctca tcgtgtctat cattgaggag gataagaact cctacacacc ctgcctcaat
     6361 cagtttcccc aggagctgaa tgtggggaag atcagtgctg aggtgatgtg gagcctgttt
     6421 gcccaggaca tgaagtacgc catggaggaa cacgacaagc accggctgtg taagagcgca
     6481 gactacatga acctgcattt caaggtcaag tggttgtaca acgagtacgt ggctgaactg
     6541 cccaccttca aggaccgcgt gccggagtac cctgcgtggt ttgagccctt cgtcatccag
     6601 tggttagatg agaatgagga ggtgtcccgg gacttcctgc atggtgcact cgagcgggac
     6661 aagaaggatg ggttccagca gacatcggag cacgccctgt tctcttgctc ggtagtggac
     6721 gtcttctccc agctcaacca gagctttgag atcatcaaga agctggagtg tcctgacccc
     6781 cagattgtgg gccactacat gcggcgcttt gccaagacca ttagcaatgt gcttctccag
     6841 tacgccgaca tcgtctccaa ggacttcgct tcctactgct ccaaggagaa ggagaaagtg
     6901 ccctgcatcc tcatgaacaa cacacagcag ctgcgggtgc agctggagaa gatgttcgag
     6961 gcgatgggtg ggaaggagct ggacgccgag gccagtggaa ccctgaagga gctgcaggtg
     7021 aaactcaaca atgtcctgga tgaactcagc cacgtgtttg ccaccagctt ccagccacac
     7081 atcgaggagt gtgtcagaca aatgggtgac atcctaagcc aagtgaaggg cacgggcaac
     7141 gtgcccgcca gtgcctgcag cagcgtggca caggacgcag acaacgtgct acagcccatc
     7201 atggatcttc tggacagcaa cctcaccctg tttgccaaaa tctgtgagaa gacggttctg
     7261 aagcgggtgc tgaaggagct gtggaagctg gtgatgaaca ccatggagag gaccattgtc
     7321 ctgccgccat ccaaactgaa ggatcacatg gtgcgagaag aagccaagag cttgaccccg
     7381 aagcagtgtg ccgtggttga actggccctg gacaccatca agcaatactt ccacgcgggg
     7441 ggcgtgggcc tcaagaagac ctttctcgag aaaagcccgg accttcagtc cctgcgctac
     7501 gccctgtcgc tctacacaca ggccaccgac ctgctcatca aaaccttcgt gcagacgcag
     7561 tcagcgcagg gctcgggtgt ggaagaccct gtaggtgaag tatccgtcca cgtggagctg
     7621 ttcacgcatc cgggaactgg ggaacagaag gtcacagtga aggtggtggc cgccaacgac
     7681 ctcaagtggc agacttctgg catcttccgt ccgttcattg aggtcaacat cgttggacct
     7741 cagctcagcg acaagaaacg aaagttcgcc accaaatcga aaaacaacag ctgggcgccc
     7801 aaatataacg agagcttcca gttctccctg agcgccgacg cgggacccga gtgctacgag
     7861 ttgcaggtgt gcgtgaagga ctactgcttc gcgcgcgagg accgcacggt ggggctggcg
     7921 gtgctgcagc tgcgggagct ggctcagcgc gggagcgccg cgtgctggct gccgctcggc
     7981 cgccgcatcc acatggacga cacggggctc acagtgctgc gtatcctgtc gcagcgcagc
     8041 aatgacgagg tggccaagga gttcgtcaag ctcaagtccg acacgcgctc ggccgaggag
     8101 ggcggtgccg cgcctgcgcc ctagaagctt gtcgagaagt actagaggat cataatcagc
     8161 cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac
     8221 ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt
     8281 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     8341 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatctg atcactgctt
     8401 gagcctagga gatccgaacc agataagtga aatctagttc caaactattt tgtcattttt
     8461 aattttcgta ttagcttacg acgctacacc cagttcccat ctattttgtc actcttccct
     8521 aaataatcct taaaaactcc atttccaccc ctcccagttc ccaactattt tgtccgccca
     8581 cagcggggca tttttcttcc tgttatgttt ttaatcaaac atcctgccaa ctccatgtga
     8641 caaaccgtca tcttcggcta ctttttctct gtcacagaat gaaaattttt ctgtcatctc
     8701 ttcgttatta atgtttgtaa ttgactgaat atcaacgctt atttgcagcc tgaatggcga
     8761 atgggacgcg ccct
//
