LOCUS       sequence-421646-        6623 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of SUMO-tagged rat syt1 (96-421).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6623)
  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 6623)
  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-syt1 (96-421)
FEATURES             Location/Qualifiers
     source          1..6623
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     694..713
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(733..810)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1016..1035
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1119..1138
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1119..1137
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1138..1162
                     /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             1177..1199
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1219..1236
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1246..1263
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1270..1563
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             2606..2623
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2676..2694)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2690..2737
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2774..3229
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2861..2880)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3071..3092
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3321..4136)
                     /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(3321..4136)
                     /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     4044..4063
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4128..4147
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4258..4846
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4747..4766
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5000..5017
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5032..5171
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5097..5116)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5257..5279
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5273..5464)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6273..6623,1..732))
                     /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 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
       61 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
      121 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
      181 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
      241 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
      301 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
      361 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
      421 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
      481 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
      541 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
      601 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
      661 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
      721 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
      781 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact
      841 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
      901 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
      961 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
     1021 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
     1081 gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga
     1141 attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta agaaggagat
     1201 atacatatgg gcagcagcca tcatcatcat catcacggca gcggcctggt gccgcgcggc
     1261 agcgctagca tgtcggactc agaagtcaat caagaagcta agccagaggt caagccagaa
     1321 gtcaagcctg agactcacat caatttaaag gtgtccgatg gatcttcaga gatcttcttc
     1381 aagatcaaaa agaccactcc tttaagaagg ctgatggaag cgttcgctaa aagacagggt
     1441 aaggaaatgg actccttaag attcttgtac gacggtatta gaattcaagc tgatcagacc
     1501 cctgaagatt tggacatgga ggataacgat attattgagg ctcacagaga acagattggt
     1561 ggatccggag gaaagaacgc cattaacatg aaagacgtga aagacttagg gaagaccatg
     1621 aaggatcagg cccttaagga tgacgatgct gaaaccggac tgactgatgg agaagaaaag
     1681 gaagagccca aggaagagga gaaactggga aagctccaat attcactgga ctatgacttc
     1741 cagaataacc agctgttggt gggaatcatc caggctgctg aactgcccgc cctggacatg
     1801 gggggtacat ccgatccata cgtcaaagtc ttcctgctgc ctgacaaaaa gaagaaattt
     1861 gagactaaag tccaccggaa aaccctcaat ccagtcttca atgaacaatt tactttcaag
     1921 gtaccctact cggaattagg tggcaaaacc ctggtgatgg ctgtgtatga ctttgatcgc
     1981 ttctccaagc acgacatcat cggagagttc aaagttccta tgaacaccgt ggattttggc
     2041 catgtgaccg aggagtggcg cgatctccag agcgctgaga aagaagagca agagaaactg
     2101 ggtgacatct gcttctccct ccgctacgtc cctactgccg gcaaactgac tgttgtcatt
     2161 ctggaagcca agaacctgaa gaagatggat gtgggtggct tatctgatcc ctacgtgaag
     2221 attcacctga tgcagaacgg taagaggctg aagaagaaaa agacgacgat taagaagaac
     2281 acactcaacc cctactacaa cgagtccttc agctttgaag ttccgttcga gcaaatccag
     2341 aaagtgcaag tggtggtaac tgttttggac tatgacaaga ttggcaagaa cgacgccatc
     2401 ggcaaagtct tcgttggtta caacagcact ggggcggagc tgcgacactg gtcagacatg
     2461 ctggccaacc cccggcgacc catcgcacag tggcacactc tgcaggtaga ggaggaggtt
     2521 gatgccatgc tggctgtcaa gaagtaaaag cttaggtatt tattcggcgc aaagtgcgtc
     2581 gggtgatgct gccaacttag tcgagcacca ccaccaccac cactgagatc cggctgctaa
     2641 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
     2701 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     2761 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     2821 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     2881 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     2941 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     3001 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     3061 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     3121 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     3181 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     3241 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     3301 atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
     3361 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
     3421 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
     3481 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
     3541 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc
     3601 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
     3661 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga
     3721 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
     3781 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca
     3841 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     3901 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     3961 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt
     4021 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     4081 atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca
     4141 ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta
     4201 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     4261 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     4321 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     4381 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     4441 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     4501 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     4561 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     4621 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     4681 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     4741 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     4801 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     4861 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     4921 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     4981 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     5041 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca
     5101 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     5161 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     5221 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     5281 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt
     5341 gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa
     5401 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg
     5461 tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac
     5521 gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt
     5581 gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc
     5641 aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg
     5701 cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg
     5761 aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc
     5821 agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc
     5881 gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca
     5941 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
     6001 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
     6061 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
     6121 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
     6181 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat
     6241 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     6301 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     6361 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
     6421 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     6481 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     6541 cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     6601 cccagcgcca tctgatcgtt ggc
//
