LOCUS       sequence-435303-        5700 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses complexin-1 in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5700)
  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 5700)
  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: CpX-1
FEATURES             Location/Qualifiers
     source          1..5700
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(578..595)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(605..622)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             641..663
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    678..702
                     /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     complement(702..721)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(703..721)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(805..824)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1030..1107
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1108..2190
                     /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(1127..1146)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2999..3190
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3184..3206)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3292..3431
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3347..3366
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3446..3463)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3617..4205)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3697..3716)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4316..4335)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4327..5142
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             4327..5142
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4400..4419)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5234..5689)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5371..5392)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5583..5602
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtcactat tacttcttga
      181 acatgtcctg cagtggccca ggcaggtact tgatgacagt gtccaggatg ctctcatctt
      241 cctcctcggg ctcgtcccca cagcctggtg ggatagcctt cttgggtcga gtcaggctgc
      301 cttctgagtt ggcctccatg gctgcctgag cctcagcctc acgctcctcc ttcttcttga
      361 tgccatactt atctcgtatg ccctgccgca tgacctcacg ttctgcctcc atcttggcat
      421 actttgcttt gcgctcctcc tctgcctgcc gcagtgcctc ctgccgctcc tcctccttct
      481 tggcggcatc ggggtccttc tcctcatcac ccccaagcat cttccccata tccttggtgg
      541 cccctcccag ggcttgtttc atcacgaact ccatatggct gccgcgcggc accaggccgc
      601 tgctgtgatg atgatgatga tggctgctgc ccatggtata tctccttctt aaagttaaac
      661 aaaattattt ctagagggga attgttatcc gctcacaatt cccctatagt gagtcgtatt
      721 aatttcgcgg gatcgagatc tcgatcctct acgccggacg catcgtggcc ggcatcaccg
      781 gcgccacagg tgcggttgct ggcgcctata tcgccgacat caccgatggg gaagatcggg
      841 ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg
      901 ccgggggact gttgggcgcc atctccttgc atgcaccatt ccttgcggcg gcggtgctca
      961 acggcctcaa cctactactg ggctgcttcc taatgcagga gtcgcataag ggagagcgtc
     1021 gagatcccgg acaccatcga atggcgcaaa acctttcgcg gtatggcatg atagcgcccg
     1081 gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag
     1141 tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct
     1201 gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc
     1261 gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg
     1321 gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt
     1381 gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg
     1441 cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag
     1501 gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct
     1561 gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg
     1621 gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct
     1681 gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag
     1741 ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa
     1801 atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg
     1861 ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg
     1921 ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag
     1981 gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag
     2041 gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg
     2101 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     2161 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtaa gttagctcac
     2221 tcattaggca ccgggatctc gaccgatgcc cttgagagcc ttcaacccag tcagctcctt
     2281 ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct ttatcatgca
     2341 actcgtagga caggtgccgg cagcgctctg ggtcattttc ggcgaggacc gctttcgctg
     2401 gagcgcgacg atgatcggcc tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca
     2461 agccttcgtc actggtcccg ccaccaaacg tttcggcgag aagcaggcca ttatcgccgg
     2521 catggcggcc ccacgggtgc gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg
     2581 gcggggttgc cttactggtt agcagaatga atcaccgata cgcgagcgaa cgtgaagcga
     2641 ctgctgctgc aaaacgtctg cgacctgagc aacaacatga atggtcttcg gtttccgtgt
     2701 ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc attatgttcc ggatctgcat
     2761 cgcaggatgc tgctggctac cctgtggaac acctacatct gtattaacga agcgctggca
     2821 ttgaccctga gtgatttttc tctggtcccg ccgcatccat accgccagtt gtttaccctc
     2881 acaacgttcc agtaaccggg catgttcatc atcagtaacc cgtatcgtga gcatcctctc
     2941 tcgtttcatc ggtatcatta cccccatgaa cagaaatccc ccttacacgg aggcatcagt
     3001 gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc agacattaac
     3061 gcttctggag aaactcaacg agctggacgc ggatgaacag gcagacatct gtgaatcgct
     3121 tcacgaccac gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga
     3181 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     3241 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat
     3301 gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag
     3361 attgtactga gagtgcacca ttgcggtgtg aaataccgca cagatgcgta aggagaaaat
     3421 accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     3481 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     3541 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     3601 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     3661 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     3721 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     3781 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     3841 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     3901 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     3961 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     4021 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     4081 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     4141 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     4201 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     4261 gttaagggat tttggtcatg aacaataaaa ctgtctgctt acataaacag taatacaagg
     4321 ggtgttatga gccatattca acgggaaacg tcttgctcta ggccgcgatt aaattccaac
     4381 atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg
     4441 acaatctatc gattgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa
     4501 ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt
     4561 atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc
     4621 actgcgatcc ccgggaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa
     4681 aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat
     4741 tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac
     4801 ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc
     4861 tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat
     4921 ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga
     4981 cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag
     5041 ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg
     5101 aataaattgc agtttcattt gatgctcgat gagtttttct aagaattaat tcatgagcgg
     5161 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     5221 aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     5281 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     5341 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     5401 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     5461 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     5521 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     5581 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     5641 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
//
