LOCUS       sequence-488332-        7446 bp    DNA     circular SYN 03-OCT-2025
DEFINITION  Calcium binding defective human Munc13-4 mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7446)
  TITLE     Martin lab plasmids
REFERENCE   2  (bases 1 to 7446)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 3, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: ptwin strep HRV3C Flag Munc13-4 C2AB
FEATURES             Location/Qualifiers
     source          1..7446
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      547..556
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(559..564,565..588,589..624,625..648)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             667..690
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             691..714
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    4174..4295
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4211..4230)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4265..4284
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4302..4757)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4439..4460)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4651..4670
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4784..4888
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4890..5247
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4890..5081
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4913..4933)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5098..5233
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5282..6076
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             5282..6076
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5336..5355)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5946..5965
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6264..6283)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6308..6355
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6684..7272
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7173..7192
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      541 ccggtcgcca ccatgggcag cgcatggagt catcctcaat tcgagaaagg tggaggttct
      601 ggcggtggat cgggaggttc agcgtggagc cacccgcagt tcgaaaaatc cggaggtgct
      661 tctggtctgg aagttctgtt ccagggaccc gattacaagg atgacgacga taagtccgga
      721 ctcagatctc gagctcaaat ggcgacactc ctctcccatc cgcagcagcg ccctcccttc
      781 ttgcgccagg ccatcaagat aaggcgccgc agagtcagag atctacagga tcccccgccc
      841 caaatggccc cggagatcca gcctccatcc caccacttct cccccgagca gcgggccctg
      901 ctctacgagg acgcactcta cactgtcttg caccgcctgg gtcatcctga gcccaaccat
      961 gtgacggagg cctctgagct gctgcgatac ctgcaggagg ccttccacgt ggagcccgag
     1021 gagcaccagc agacactgca gcgggtcagg gagcttgaga agccaatatt ttgtctgaag
     1081 gcaacagtga aacaggccaa gggcattctg ggcaaaaatg tcagtgggtt cagcaacccc
     1141 tactgcctgc tgggcattga gcagggggta ggtgtgccag ggggcagccc cgggtcccgg
     1201 catcggcaga aggctgtggt gaggcacacc atccccgagg aggagaccca ccgcacgcag
     1261 gtcatcaccc agacactcaa ccccgtctgg gacgagacct tcatcctgga gtttgaggac
     1321 atcaccaatg cgagctttca tctggacatg tgggacctgg acactgtgga gtctgtccga
     1381 cagaagcttg gggagctcac ggatctgcat gggcttcgca ggatctttaa agaggcccgg
     1441 aaggacaaag gccaggacga ctttctgggg aacgtggttc tgaggctgca ggacctgcgc
     1501 tgccgagagg accagtggta ccccctggaa ccccgcactg agacctaccc agaccgaggc
     1561 cagtgccacc tccagttcca actcatccat aagcggagag ccacttcggc cagccgctcg
     1621 cagcccagct acaccgtgca cctccacctc ctgcagcagc ttgtgtccca cgaggtcacc
     1681 cagcacgagg cgggaagcac ctcctgggac gggtcgctga gtccccaggc tgccaccgtc
     1741 ctctttctgc acgccacaca gaaggaccta tccgacttcc accagtccat ggcgcagtgg
     1801 ctggcctaca gccgcctcta ccagagcctg gagttcccca gcagctgcct cctgcacccc
     1861 atcaccagca tcgagtacca gtggatccag ggtcggctca aggcagaaca gcaggaggag
     1921 ctggccgcct cattcagctc cctgctgacc tacggcctct ccctcatccg gaggttccgc
     1981 tctgtcttcc ccctctctgt ctcggactcc ccagcccggc tgcagtctct tctcagggtc
     2041 ctggtacaga tgtgcaagat gaaggccttt ggagaactgt gccccaacac cgccccattg
     2101 ccccagctgg tgactgaggc cctgcagact ggcaccactg aatggttcca cctgaagcag
     2161 cagcaccatc aacccatggt gcagggcatc ccggaggcag gcaaggcctt gctgggcctg
     2221 gtacaggatg tcattggcga cctgcaccag tgccagcgca catgggacaa gatcttccac
     2281 aataccctca agatccacct cttctccatg gctttccggg agctgcagtg gctggtggcc
     2341 aagcgggtgc aggaccacac gacggttgtg ggtgatgtag tgtccccaga gatgggcgag
     2401 agtctgttcc agctctacat cagcctcaag gagctctgcc agctgcgcat gagctcctca
     2461 gagagggatg gagtcctggc cctggataat ttccaccgct ggttccagcc ggccatcccc
     2521 tcctggctgc agaagacgta caacgaggcc ctggcgcggg tgcagcgcgc tgtgcagatg
     2581 gatgagctgg tgcccctggg tgaactgacc aagcacagca catccgcagt agacctctca
     2641 acctgctttg cccagatcag ccacactgcc cggcagctgg actggccaga cccagaggag
     2701 gccttcatga ttaccgtcaa gtttgtggag gacacctgtc gcctggccct ggtgtactgc
     2761 agccttataa aggcccgggc ccgcgagctc tcttcaggcc agaaggacca aggccaggca
     2821 gccaacatgc tgtgtgtggt ggtgaatgac atggagcagc tgcggctggt gatcggcaag
     2881 ttgcccgccc agctggcatg ggaggccctg gagcagcggg taggggccgt gctggagcag
     2941 gggcagctgc agaacacgct gcatgcccag ctgcagagcg cgctggccgg gctgggccat
     3001 gagatccgca ctggcgtccg caccctggcc gagcagttgg aggtgggcat cgccaagcac
     3061 atccagaaac tggtgggcgt cagggagtct gtcctgcctg aggatgccat tctgcccctg
     3121 atgaagttcc tggaggtgga gctttgctac atgaacacca acttggtgca ggaaaacttc
     3181 agcagcctcc tgaccctgct ctggacccac acactcacag tgctggtgga ggcggccgcc
     3241 tcccagcgca gctcatccct ggcttccaac aggctgaaga ttgccctgca gaacctggag
     3301 atctgcttcc acgctgaggg ctgtggcctg ccacccgagg ccctgcacac tgccaccttc
     3361 caggctctgc agagggacct ggagctgcag gcggcctcca gccgggaact catccggaag
     3421 tacttctgca gccgaatcca gcagcaggca gaaaccacct ctgaggagct gggggctgtg
     3481 acagtcaagg cctcctaccg cgcctctgag cagaagctgc gtgtggagct gctcagcgcc
     3541 tccagcctgc tgcccctgaa ctccaatggc tccagcaacc cctttgtcca gctgaccttg
     3601 gagcccaggc atgagttccc tgagctggcc gcccgggaga cccagaagca caagaaggac
     3661 cttcacccat tgtttgatga gacctttgaa ttcctggtgc ctgctgagcc gtgccgcaag
     3721 gctggggcat gcctcctgct caccgtgctg gactacgaca cgctgggggc cgacgacctg
     3781 gaaggcgagg ccttcctgcc gctgcgtgag gtgcccgggc tgagtggctc tgaggagcct
     3841 ggtgaggtgc ctcagacccg cctgcccctc acgtaccccg cacccaacgg ggacccaatc
     3901 ctgcagctgc tggagggccg gaagggtgac cgagaggccc aggtctttgt gaggctgcgg
     3961 cggcaccggg ccaagcaggc ctcccagcat gccttgcggc cggcaccgta atcgaattct
     4021 gcagtcgacg gtaccgcggg cccgggatcc accggatcta gataactgat cataatcagc
     4081 cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac
     4141 ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt
     4201 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     4261 agttgtggtt tgtccaaact catcaatgta tcttaacgcg taaattgtaa gcgttaatat
     4321 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     4381 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     4441 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     4501 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     4561 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     4621 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     4681 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     4741 gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     4801 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     4861 gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg
     4921 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     4981 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     5041 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     5101 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     5161 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
     5221 ttttttggag gcctaggctt ttgcaaagat cgatcaagag acaggatgag gatcgtttcg
     5281 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     5341 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     5401 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     5461 gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     5521 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     5581 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     5641 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     5701 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     5761 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga
     5821 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     5881 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     5941 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     6001 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     6061 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     6121 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     6181 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     6241 gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc
     6301 gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa
     6361 cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc
     6421 caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca
     6481 gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata
     6541 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     6601 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     6661 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     6721 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     6781 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     6841 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     6901 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     6961 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     7021 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     7081 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     7141 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     7201 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     7261 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     7321 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     7381 atgcattagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
     7441 gttccg
//
