LOCUS       sequence-421656-        6449 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of SUMO-tagged mouse syt7 C2AB - 4A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6449)
  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 6449)
  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 C2AB - 4A
FEATURES             Location/Qualifiers
     source          1..6449
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     781..800
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(820..897)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1103..1122
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1206..1225
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1206..1224
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1225..1249
                     /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             1264..1286
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1306..1323
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1333..1350
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1357..1650
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             2519..2536
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2589..2607)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2603..2650
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2687..3142
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2774..2793)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2984..3005
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3234..4049)
                     /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(3234..4049)
                     /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     3957..3976
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4041..4060
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4171..4759
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4660..4679
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4913..4930
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4945..5084
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5010..5029)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5170..5192
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5186..5377)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6186..6449,1..819))
                     /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 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
       61 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
      121 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
      181 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
      241 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
      301 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
      361 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
      421 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
      481 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
      541 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
      601 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
      661 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
      721 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
      781 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
      841 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
      901 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
      961 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     1021 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     1081 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     1141 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc
     1201 gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat
     1261 aattttgttt aactttaaga aggagatata catatgggca gcagccatca tcatcatcat
     1321 cacggcagcg gcctggtgcc gcgcggcagc gctagcatgt cggactcaga agtcaatcaa
     1381 gaagctaagc cagaggtcaa gccagaagtc aagcctgaga ctcacatcaa tttaaaggtg
     1441 tccgatggat cttcagagat cttcttcaag atcaaaaaga ccactccttt aagaaggctg
     1501 atggaagcgt tcgctaaaag acagggtaag gaaatggact ccttaagatt cttgtacgac
     1561 ggtattagaa ttcaagctga tcagacccct gaagatttgg acatggagga taacgatatt
     1621 attgaggctc acagagaaca gattggtgga tccatgggcc gaatccagtt cagtgttggc
     1681 tacaacttcc aagagtccac actcaccgtg aaggtcatga aggcccaaga gctgccagcc
     1741 aaggacgcca gcggtactag tgaccccttt gtcaagatct acctgctacc cgacaagaag
     1801 cacaaactgg agaccaaggt gaagcggaag aatctaaacc cgcactggaa tgagaccttt
     1861 ctatttgaag ggtttcccta cgagaaagtg gtgcagaggg tcctctacct ccaggtcctg
     1921 gattatgacc gtgcaagccg caatgacccc attggggagg tgtccatccc tctgaacaag
     1981 gtggacctga cccagatgca gaccttctgg aaggatctga agccatgcag cgatgggagt
     2041 gggagccgag gggagctgct cttgtccctc tgctacaacc cctctgccaa ctccatcatc
     2101 gtgaacatca tcaaagctcg aaacctcaaa gccatggacg cagggggcac atcagacccc
     2161 tatgtgaagg tgtggctgat gtataaagac aagcgggtag agaaaaagaa gaccgtgaca
     2221 aagaagagga acctgaaccc catcttcaat gagtctttcg ccttcgacat acccacggag
     2281 aagctgaggg agaccacgat catcatcact gtcatggaca aagacaaggc tagccgcaac
     2341 gacgtcatcg gcaagatcta cctgtcctgg aagagcggac caggtgaagt gaaacactgg
     2401 aaggacatga tcgctcgtcc ccggcagcct gtggcccagt ggcaccagct gaaagcctga
     2461 aagcttaggt atttattcgg cgcaaagtgc gtcgggtgat gctgccaact tagtcgagca
     2521 ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc
     2581 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     2641 gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc
     2701 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     2761 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     2821 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     2881 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     2941 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     3001 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     3061 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     3121 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     3181 tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa
     3241 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat
     3301 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg
     3361 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat
     3421 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc
     3481 ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta
     3541 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
     3601 gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac
     3661 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct
     3721 aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga
     3781 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
     3841 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct
     3901 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg
     3961 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag
     4021 caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca
     4081 gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     4141 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     4201 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     4261 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     4321 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     4381 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     4441 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     4501 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     4561 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     4621 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     4681 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     4741 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     4801 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     4861 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     4921 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     4981 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     5041 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     5101 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     5161 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     5221 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     5281 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     5341 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     5401 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     5461 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     5521 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     5581 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     5641 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     5701 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     5761 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     5821 ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc
     5881 cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga
     5941 ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa
     6001 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa
     6061 gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     6121 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     6181 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     6241 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     6301 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     6361 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     6421 atataacatg agctgtcttc ggtatcgtc
//
