LOCUS       sequence-421648-        6455 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of SUMO-tagged mouse syt7 (136-403) L361C .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6455)
  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 6455)
  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 (136-403) L361C
FEATURES             Location/Qualifiers
     source          1..6455
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     172..193
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(422..1237)
                     /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(422..1237)
                     /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     1145..1164
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1229..1248
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1359..1947
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1848..1867
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2101..2118
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2133..2272
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2198..2217)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2358..2380
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2374..2565)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3374..4456)
                     /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     4418..4437
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4457..4534)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4740..4759
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4843..4862
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4843..4861
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4862..4886
                     /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             4901..4923
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4943..4960
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4970..4987
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             4994..5287
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             6162..6179
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6232..6250)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6246..6293
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      join(6330..6455,1..330)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6417..6436)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
       61 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
      121 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
      181 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
      241 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
      301 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
      361 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt
      421 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
      481 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
      541 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
      601 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
      661 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
      721 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
      781 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
      841 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
      901 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
      961 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     1021 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     1081 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     1141 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     1201 gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta
     1261 tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac
     1321 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     1381 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     1441 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     1501 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     1561 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     1621 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     1681 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     1741 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     1801 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     1861 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     1921 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     1981 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     2041 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     2101 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     2161 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     2221 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     2281 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     2341 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     2401 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     2461 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     2521 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     2581 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     2641 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     2701 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     2761 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     2821 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     2881 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     2941 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     3001 caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg
     3061 cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa
     3121 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc
     3181 gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac
     3241 agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
     3301 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     3361 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     3421 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     3481 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
     3541 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
     3601 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg
     3661 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
     3721 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa
     3781 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
     3841 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
     3901 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
     3961 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
     4021 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
     4081 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
     4141 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
     4201 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
     4261 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
     4321 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
     4381 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
     4441 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
     4501 taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc
     4561 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
     4621 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
     4681 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
     4741 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
     4801 atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag
     4861 gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg
     4921 agatatacat atgggcagca gccatcatca tcatcatcac ggcagcggcc tggtgccgcg
     4981 cggcagcgct agcatgtcgg actcagaagt caatcaagaa gctaagccag aggtcaagcc
     5041 agaagtcaag cctgagactc acatcaattt aaaggtgtcc gatggatctt cagagatctt
     5101 cttcaagatc aaaaagacca ctcctttaag aaggctgatg gaagcgttcg ctaaaagaca
     5161 gggtaaggaa atggactcct taagattctt gtacgacggt attagaattc aagctgatca
     5221 gacccctgaa gatttggaca tggaggataa cgatattatt gaggctcaca gagaacagat
     5281 tggtggatcc gagaacctgg gccgaatcca gttcagtgtt ggctacaact tccaagagtc
     5341 cacactcacc gtgaaggtca tgaaggccca agagctgcca gccaaggact tcagcggtac
     5401 tagtgacccc tttgtcaaga tctacctgct acccgacaag aagcacaaac tggagaccaa
     5461 ggtgaagcgg aagaatctaa acccgcactg gaatgagacc tttctatttg aagggtttcc
     5521 ctacgagaaa gtggtgcaga gggtcctcta cctccaggtc ctggattatg accgtttcag
     5581 ccgcaatgac cccattgggg aggtgtccat ccctctgaac aaggtggacc tgacccagat
     5641 gcagaccttc tggaaggatc tgaagccagc cagcgatggg agtgggagcc gaggggagct
     5701 gctcttgtcc ctcgcctaca acccctctgc caactccatc atcgtgaaca tcatcaaagc
     5761 tcgaaacctc aaagccatgg acatcggggg cacatcagac ccctatgtga aggtgtggct
     5821 gatgtataaa gacaagcggg tagagaaaaa gaagactgtg acaaagaaga ggaacctgaa
     5881 ccccatcttc aatgagtctt tcgccttcga catacccacg gagaagctga gggagaccac
     5941 gatcatcatc actgtcatgg acaaagacaa gtgcagccgc aacgacgtca tcggcaagat
     6001 ctacctgtcc tggaagagcg gaccaggtga agtgaaacac tggaaggaca tgatcgctcg
     6061 tccccggcag cctgtggccc agtggcacca gctgaaagcc tgaaagctta ggtatttatt
     6121 cggcgcaaag tgcgtcgggt gatgctgcca acttagtcga gcaccaccac caccaccact
     6181 gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc
     6241 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     6301 gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     6361 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     6421 tttcgctttc ttcccttcct ttctcgccac gttcg
//
