LOCUS       sequence-421659-        6905 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of SUMO-tagged rat FL-syt1 5W.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6905)
  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 6905)
  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-syt1 5W
FEATURES             Location/Qualifiers
     source          1..6905
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      39..627
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     528..547
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     781..798
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    813..952
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(878..897)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1038..1060
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1054..1245)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2054..3136)
                     /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     3098..3117
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3137..3214)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3420..3439
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3523..3542
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3523..3541
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3542..3566
                     /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             3581..3603
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3623..3640
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3650..3667
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3674..3967
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             5292..5309
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5362..5380)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5376..5423
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5460..5915
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5547..5566)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5757..5778
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6007..6822)
                     /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(6007..6822)
                     /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     6730..6749
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6814..6833
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
       61 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
      121 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
      181 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
      241 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
      301 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
      361 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
      421 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
      481 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
      541 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
      601 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
      661 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
      721 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
      781 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
      841 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
      901 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
      961 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     1021 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     1081 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     1141 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     1201 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     1261 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     1321 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     1381 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     1441 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     1501 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     1561 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     1621 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     1681 caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg
     1741 cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa
     1801 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc
     1861 gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac
     1921 agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
     1981 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     2041 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     2101 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     2161 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
     2221 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
     2281 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg
     2341 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
     2401 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa
     2461 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
     2521 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
     2581 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
     2641 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
     2701 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
     2761 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
     2821 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
     2881 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
     2941 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
     3001 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
     3061 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
     3121 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
     3181 taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc
     3241 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
     3301 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
     3361 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
     3421 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
     3481 atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag
     3541 gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg
     3601 agatatacat atgggcagca gccatcatca tcatcatcac ggcagcggcc tggtgccgcg
     3661 cggcagcgct agcatgtcgg actcagaagt caatcaagaa gctaagccag aggtcaagcc
     3721 agaagtcaag cctgagactc acatcaattt aaaggtgtcc gatggatctt cagagatctt
     3781 cttcaagatc aaaaagacca ctcctttaag aaggctgatg gaagcgttcg ctaaaagaca
     3841 gggtaaggaa atggactcct taagattctt gtacgacggt attagaattc aagctgatca
     3901 gacccctgaa gatttggaca tggaggataa cgatattatt gaggctcaca gagaacagat
     3961 tggtggatcc gtgagtgcca gtcatcctga ggccctggcc gcccctgtca ccactgttgc
     4021 gacccttgtc ccacacaatg ccactgagcc agccagtcct ggggaaggga aggaagatgc
     4081 cttttccaag ctgaagcaga agtttatgaa tgagctgcat aaaattccat tgccaccgtg
     4141 ggccttaata gccatagcca tagttgcggt ccttttagtc gtaacctgct gcttttgtgt
     4201 ctgtaagaaa tgtttgttca aaaagaaaaa caagaagaag gggaaggaaa agggaggaaa
     4261 gaacgccatt aacatgaaag acgtgaaaga cttagggaag accatgaagg atcaggccct
     4321 taaggatgac gatgctgaaa ccggactgac tgatggagaa gaaaaggaag agcccaagga
     4381 agaggagaag cttggaaagc tccaatattc actggactat gacttccaga ataaccagct
     4441 gttggtggga atcatccagg ctgctgaact gcccgccctg gactgggggg gtacatccga
     4501 tccatacgtc aaagtcttcc tgctgcctga caaaaagaag aaatttgaga ctaaagtcca
     4561 ccggaaaacc ctcaatccag tcttcaatga acaatttact ttcaaggtac cctactcgga
     4621 attaggtggc aaaaccctgg tgatggctgt gtatgactgg gatcgctggt ccaagcacga
     4681 catcatcgga gagttcaaag ttcctatgaa caccgtggat tttggccatg tgaccgagga
     4741 gtggcgcgat ctccagagcg ctgagaaaga agagcaagag aaactgggtg acatcgcatt
     4801 ctccctccgc tacgtcccta ctgccggcaa actgactgtt gtcattctgg aagccaagaa
     4861 cctgaagaag atggattggg gtggcttatc tgatccctac gtgaagattc acctgatgca
     4921 gaacggtaag aggctgaaga agaaaaagac gacgattaag aagaacacac tcaaccccta
     4981 ctacaacgag tccttcagct ttgaagttcc gttcgagcaa atccagaaag tgcaagtggt
     5041 ggtaactgtt ttggactggg acaagtgcgg caagaacgac gcgatcggca aagtcttcgt
     5101 tggttacaac agcactgggg cggagctgcg acactggtca gacatgctgg ccaacccccg
     5161 gcgacccatc gcacagtggc acactctgca ggtagaggag gaggttgatg ccatgctggc
     5221 tgtcaagaag taaaagctta ggtatttatt cggcgcaaag tgcgtcgggt gatgctgcca
     5281 acttagtcga gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg
     5341 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     5401 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga
     5461 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     5521 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     5581 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     5641 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     5701 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     5761 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     5821 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     5881 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
     5941 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat
     6001 taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
     6061 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
     6121 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
     6181 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
     6241 gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac
     6301 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
     6361 tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg
     6421 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
     6481 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca
     6541 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     6601 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     6661 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg
     6721 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     6781 tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact
     6841 gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt
     6901 tccac
//
