LOCUS       sequence-421658-        6623 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of SUMO-tagged rat syt1 s7+.
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 s7+
FEATURES             Location/Qualifiers
     source          1..6623
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     91..110
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     175..194
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      305..893
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     794..813
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1047..1064
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1079..1218
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1144..1163)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1304..1326
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1320..1511)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2320..3402)
                     /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     3364..3383
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3403..3480)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3686..3705
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3789..3808
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3789..3807
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3808..3832
                     /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             3847..3869
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3889..3906
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3916..3933
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3940..4233
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             5276..5293
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5346..5364)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5360..5407
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5444..5899
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5531..5550)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5741..5762
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(5991..6623,1..183))
                     /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(join(5991..6623,1..183))
                     /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"
ORIGIN
        1 aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg
       61 atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc
      121 agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct
      181 cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc atgaccaaaa
      241 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
      301 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
      361 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
      421 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
      481 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
      541 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
      601 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
      661 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
      721 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
      781 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
      841 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
      901 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
      961 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     1021 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     1081 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct
     1141 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
     1201 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     1261 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     1321 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg
     1381 tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc
     1441 tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc
     1501 tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg
     1561 atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg
     1621 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact
     1681 cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag
     1741 catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga
     1801 ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg
     1861 cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg
     1921 caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg
     1981 gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg
     2041 acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc
     2101 gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt
     2161 cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc
     2221 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     2281 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     2341 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     2401 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     2461 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     2521 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     2581 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg
     2641 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
     2701 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     2761 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     2821 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     2881 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
     2941 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     3001 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
     3061 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     3121 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     3181 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     3241 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     3301 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     3361 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     3421 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     3481 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
     3541 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
     3601 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
     3661 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
     3721 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc
     3781 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
     3841 aataattttg tttaacttta agaaggagat atacatatgg gcagcagcca tcatcatcat
     3901 catcacggca gcggcctggt gccgcgcggc agcgctagca tgtcggactc agaagtcaat
     3961 caagaagcta agccagaggt caagccagaa gtcaagcctg agactcacat caatttaaag
     4021 gtgtccgatg gatcttcaga gatcttcttc aagatcaaaa agaccactcc tttaagaagg
     4081 ctgatggaag cgttcgctaa aagacagggt aaggaaatgg actccttaag attcttgtac
     4141 gacggtatta gaattcaagc tgatcagacc cctgaagatt tggacatgga ggataacgat
     4201 attattgagg ctcacagaga acagattggt ggatccggag gaaagaacgc cattaacatg
     4261 aaagacgtga aagacttagg gaagaccatg aaggatcagg cccttaagga tgacgatgct
     4321 gaaaccggac tgactgatgg agaagaaaag gaagagccca aggaagagga gaaactggga
     4381 aagctccaat attcactgga ctatgacttc cagaataacc agctgttggt gggaatcatc
     4441 caggctgctg aactgcccgc caaggacatg gggggtacat ccgatccata cgtcaaagtc
     4501 ttcctgctgc ctgacaaaaa gaagaaattt gagactaaag tcaagcggaa aaccctcaat
     4561 ccagtcttca atgaacaatt tactttcaag gtgccctact cggaattagg tggcaaaacc
     4621 ctggtgatgg ctgtgtatga ctttgatcgc ttctccaggc atgacatcat cggagagttc
     4681 aaagttccta tgaacaccgt ggattttggc catgtgaccg aggagtggcg tgatctccag
     4741 agcgctgaga aagaagagca agagaaactg ggtgacatcg ccttctccct ccgctacgtc
     4801 cctactgccg gcaaactgac tgttgtcatt ctggaagcca agaacctgaa gaagatggat
     4861 gtgggtggct tatctgatcc ctacgtgaag attcacctga tgcagaacgg taagaggctg
     4921 aagaagaaaa agacgacgat taagaagagg acactcaacc cctactacaa cgagtccttc
     4981 agctttgaag ttccgttcga gcaaatccag aaagtgcaag tggtggtaac tgttttggac
     5041 aaggacaagt gtggcaggaa cgacgccatc ggcaaagtct tcgttggtta caacagcact
     5101 ggggcggagc tgcgacactg gtcagacatg ctggccaacc cccggcgacc catcgcacag
     5161 tggcacactc tgcaggtaga ggaggaggtt gatgccatgc tggctgtcaa gaagtaaaag
     5221 cttaggtatt tattcggcgc aaagtgcgtc gggtgatgct gccaacttag tcgagcacca
     5281 ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc
     5341 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     5401 ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc
     5461 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     5521 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     5581 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     5641 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     5701 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     5761 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     5821 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     5881 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     5941 gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac
     6001 tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt
     6061 tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca
     6121 agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc
     6181 ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt
     6241 gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc
     6301 tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg
     6361 agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg
     6421 cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat
     6481 acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta
     6541 cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc
     6601 atctcatctg taacatcatt ggc
//
