LOCUS       sequence-421650-        6854 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of SUMO-tagged mouse FL-syt7.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6854)
  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 6854)
  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-FL-syt7
FEATURES             Location/Qualifiers
     source          1..6854
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     578..597
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(617..694)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     900..919
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1003..1022
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1003..1021
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1022..1046
                     /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             1061..1083
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1103..1120
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1130..1147
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1154..1447
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             2721..2738
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2791..2809)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2805..2852
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2889..3344
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2976..2995)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3186..3207
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3436..4251)
                     /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(3436..4251)
                     /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     4159..4178
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4243..4262
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4373..4961
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4862..4881
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5115..5132
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5147..5286
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5212..5231)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5372..5394
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5388..5579)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6388..6854,1..616))
                     /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 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
       61 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
      121 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
      181 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
      241 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
      301 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
      361 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
      421 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
      481 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
      541 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
      601 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
      661 taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc
      721 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
      781 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
      841 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
      901 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
      961 atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag
     1021 gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg
     1081 agatatacat atgggcagca gccatcatca tcatcatcac ggcagcggcc tggtgccgcg
     1141 cggcagcgct agcatgtcgg actcagaagt caatcaagaa gctaagccag aggtcaagcc
     1201 agaagtcaag cctgagactc acatcaattt aaaggtgtcc gatggatctt cagagatctt
     1261 cttcaagatc aaaaagacca ctcctttaag aaggctgatg gaagcgttcg ctaaaagaca
     1321 gggtaaggaa atggactcct taagattctt gtacgacggt attagaattc aagctgatca
     1381 gacccctgaa gatttggaca tggaggataa cgatattatt gaggctcaca gagaacagat
     1441 tggtggatcc atgtaccggg acccggaggc ggccagccca ggggcaccta cccgcgatgt
     1501 cctgctggtc tctgcaatca tcaccgtcag ccttagcgtc actatcgtcc tctgcggcct
     1561 gtgccactgg tgtcagcgca aactgggcaa acgctacaag aattccttgg agacggtggg
     1621 cacgccagac tcggggcgtg ggcgcggtga gaagaaagcc atcaagttgc ctgcaggagg
     1681 gaaggctgtg aatacagccc cagtgcccgg ccagacgcca cacgatgagt ctgaccgcag
     1741 aacggagacc cgttcctctg tctcggacct cgtcaactcc cttaccagcg agatgctcat
     1801 gctctcccca ggttctgagg aggatgaggc ccacgagggc tgcagccgag agaacctggg
     1861 ccgaatccag ttcagtgttg gctacaactt ccaagagtcc acactcaccg tgaaggtcat
     1921 gaaggcccaa gagctgccag ccaaggactt cagcggtact agtgacccct ttgtcaagat
     1981 ctacctgcta cccgacaaga agcacaaact ggagaccaag gtgaagcgga agaatctaaa
     2041 cccgcactgg aatgagacct ttctatttga agggtttccc tacgagaaag tggtgcagag
     2101 ggtcctctac ctccaggtcc tggattatga ccgtttcagc cgcaatgacc ccattgggga
     2161 ggtgtccatc cctctgaaca aggtggacct gacccagatg cagaccttct ggaaggatct
     2221 gaagccatgc agcgatggga gtgggagccg aggggagctg ctcttgtccc tctgctacaa
     2281 cccctctgcc aactccatca tcgtgaacat catcaaagct cgaaacctca aagccatgga
     2341 catcgggggc acatcagacc cctatgtgaa ggtgtggctg atgtataaag acaagcgggt
     2401 agagaaaaag aagactgtga caaagaagag gaacctgaac cccatcttca atgagtcttt
     2461 cgccttcgac atacccacgg agaagctgag ggagaccacg atcatcatca ctgtcatgga
     2521 caaagacaag ctcagccgca acgacgtcat cggcaagatc tacctgtcct ggaagagcgg
     2581 accaggtgaa gtgaaacact ggaaggacat gatcgctcgt ccccggcagc ctgtggccca
     2641 gtggcaccag ctgaaagcct gaaagcttag gtatttattc ggcgcaaagt gcgtcgggtg
     2701 atgctgccaa cttagtcgag caccaccacc accaccactg agatccggct gctaacaaag
     2761 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     2821 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg
     2881 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     2941 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     3001 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     3061 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     3121 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     3181 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     3241 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     3301 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     3361 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     3421 ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
     3481 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     3541 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
     3601 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     3661 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac
     3721 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
     3781 attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt
     3841 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     3901 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
     3961 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     4021 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     4081 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
     4141 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     4201 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct
     4261 tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg
     4321 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     4381 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     4441 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     4501 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     4561 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     4621 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     4681 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     4741 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     4801 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     4861 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     4921 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     4981 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     5041 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     5101 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     5161 ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg
     5221 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     5281 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     5341 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     5401 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     5461 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     5521 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     5581 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     5641 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     5701 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     5761 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     5821 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     5881 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     5941 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     6001 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg
     6061 gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga
     6121 gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag
     6181 cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc
     6241 atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag
     6301 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     6361 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     6421 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     6481 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     6541 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     6601 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     6661 taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     6721 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     6781 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     6841 aatttgattg cgag
//
