LOCUS       sequence-421653-        6448 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of SUMO-tagged mouse syt7 C2AB-2A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6448)
  AUTHORS   Courtney KC, Mandal T, Mehta N, Wu L, Li Y, Das D, Cui Q, Chapman ER
  TITLE     Synaptotagmin-7 outperforms synaptotagmin-1 to promote the formation
            of large, stable fusion pores via robust membrane penetration.
  JOURNAL   Nat Commun. 2023 Nov 27;14(1):7761. doi: 10.1038/s41467-023-42497-8.
  PUBMED    38012142
REFERENCE   2  (bases 1 to 6448)
  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 
            Commun."; date: "2023-11-27"; volume: "14(1):7761. doi"; pages: " 
            10.1038/s41467-023-42497-8"
COMMENT     Sequence Label: pET SUMO-syt7 C2AB-2A
FEATURES             Location/Qualifiers
     source          1..6448
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     781..800
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(820..897)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1103..1122
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1206..1225
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1206..1224
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1225..1249
                     /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             1264..1286
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1306..1323
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1333..1350
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1357..1650
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             2518..2535
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2588..2606)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2602..2649
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2686..3141
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2773..2792)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2983..3004
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3233..4048)
                     /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             complement(3233..4048)
                     /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     3956..3975
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4040..4059
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4170..4758
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4659..4678
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4912..4929
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4944..5083
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5009..5028)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5169..5191
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5185..5376)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6185..6448,1..819))
                     /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"
ORIGIN
        1 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
       61 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
      121 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
      181 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
      241 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
      301 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
      361 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
      421 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
      481 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
      541 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
      601 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
      661 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
      721 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
      781 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
      841 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
      901 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
      961 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     1021 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     1081 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     1141 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc
     1201 gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat
     1261 aattttgttt aactttaaga aggagatata catatgggca gcagccatca tcatcatcat
     1321 cacggcagcg gcctggtgcc gcgcggcagc gctagcatgt cggactcaga agtcaatcaa
     1381 gaagctaagc cagaggtcaa gccagaagtc aagcctgaga ctcacatcaa tttaaaggtg
     1441 tccgatggat cttcagagat cttcttcaag atcaaaaaga ccactccttt aagaaggctg
     1501 atggaagcgt tcgctaaaag acagggtaag gaaatggact ccttaagatt cttgtacgac
     1561 ggtattagaa ttcaagctga tcagacccct gaagatttgg acatggagga taacgatatt
     1621 attgaggctc acagagaaca gattggtgga tccatgggcc gaatccagtt cagtgttggc
     1681 tacaacttcc aagagtccac actcaccgtg aaggtcatga aggcccaaga gctgccagcc
     1741 aaggacttca gcggtactag tgaccccttt gtcaagatct acctgctacc cgacaagaag
     1801 cacaaactgg agaccaaggt gaagcggaag aatctaaacc cgcactggaa tgagaccttt
     1861 ctatttgaag ggtttcccta cgagaaagtg gtgcagaggg tcctctacct ccaggtcctg
     1921 gattatgacc gtttcagccg caatgacccc attggggagg tgtccatccc tctgaacaag
     1981 gtggacctga cccagatgca gaccttctgg aaggatctga agccatgcag cgatgggagt
     2041 gggagccgag gggagctgct cttgtccctc tgctacaacc cctctgccaa ctccatcatc
     2101 gtgaacatca tcaaagctcg aaacctcaaa gccatggacg cagggggcac atcagacccc
     2161 tatgtgaagg tgtggctgat gtataaagac aagcgggtag agaaaaagaa gaccgtgaca
     2221 aagaagagga acctgaaccc catcttcaat gagtctttcg ccttcgacat acccacggag
     2281 aagctgaggg agaccacgat catcatcact gtcatggaca aagacaaggc tagccgcaac
     2341 gacgtcatcg gcaagatcta cctgtcctgg aagagcggac caggtgaagt gaaacactgg
     2401 aaggacatga tcgctcgtcc ccggcagcct gtggcccagt ggcaccagct gaaagcctga
     2461 agcttaggta tttattcggc gcaaagtgcg tcgggtgatg ctgccaactt agtcgagcac
     2521 caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct
     2581 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     2641 ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg
     2701 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     2761 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     2821 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     2881 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     2941 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     3001 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     3061 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     3121 aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat
     3181 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa
     3241 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
     3301 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
     3361 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
     3421 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
     3481 gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac
     3541 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
     3601 cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
     3661 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta
     3721 atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag
     3781 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
     3841 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
     3901 gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc
     3961 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc
     4021 aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag
     4081 acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     4141 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     4201 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     4261 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     4321 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     4381 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     4441 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     4501 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     4561 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     4621 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     4681 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     4741 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     4801 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     4861 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     4921 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     4981 atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     5041 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     5101 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     5161 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     5221 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     5281 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     5341 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     5401 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     5461 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     5521 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     5581 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     5641 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     5701 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     5761 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     5821 gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc
     5881 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     5941 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     6001 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     6061 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     6121 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     6181 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     6241 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     6301 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     6361 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     6421 tataacatga gctgtcttcg gtatcgtc
//
