LOCUS       sequence-421662-        6449 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of SUMO-tagged mouse syt7 (136-403).
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 (136-403)
FEATURES             Location/Qualifiers
     source          1..6449
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      92..680
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     581..600
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     834..851
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    866..1005
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(931..950)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1091..1113
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1107..1298)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2107..3189)
                     /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     3151..3170
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3190..3267)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3473..3492
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3576..3595
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3576..3594
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3595..3619
                     /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             3634..3656
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3676..3693
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3703..3720
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3727..4020
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             4889..4906
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4959..4977)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4973..5020
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5057..5512
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5144..5163)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5354..5375
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5604..6419)
                     /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(5604..6419)
                     /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     6327..6346
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6411..6430
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
       61 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
      121 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
      181 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
      241 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
      301 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
      361 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
      421 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
      481 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
      541 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
      601 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
      661 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
      721 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
      781 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
      841 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
      901 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
      961 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     1021 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     1081 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     1141 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     1201 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     1261 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     1321 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     1381 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     1441 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     1501 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     1561 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     1621 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     1681 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     1741 aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg
     1801 ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg
     1861 aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa
     1921 atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata
     1981 agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     2041 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     2101 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     2161 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     2221 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     2281 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     2341 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac
     2401 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     2461 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     2521 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     2581 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     2641 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     2701 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     2761 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     2821 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     2881 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     2941 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     3001 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     3061 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     3121 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     3181 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     3241 aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct
     3301 gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg
     3361 gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca
     3421 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt
     3481 cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc
     3541 cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta taggggaatt
     3601 gtgagcggat aacaattccc ctctagaaat aattttgttt aactttaaga aggagatata
     3661 catatgggca gcagccatca tcatcatcat cacggcagcg gcctggtgcc gcgcggcagc
     3721 gctagcatgt cggactcaga agtcaatcaa gaagctaagc cagaggtcaa gccagaagtc
     3781 aagcctgaga ctcacatcaa tttaaaggtg tccgatggat cttcagagat cttcttcaag
     3841 atcaaaaaga ccactccttt aagaaggctg atggaagcgt tcgctaaaag acagggtaag
     3901 gaaatggact ccttaagatt cttgtacgac ggtattagaa ttcaagctga tcagacccct
     3961 gaagatttgg acatggagga taacgatatt attgaggctc acagagaaca gattggtgga
     4021 tccctgggcc gaatccagtt cagtgttggc tacaacttcc aagagtccac actcaccgtg
     4081 aaggtcatga aggcccaaga gctgccagcc aaggacttca gcggtactag tgaccccttt
     4141 gtcaagatct acctgctacc cgacaagaag cacaaactgg agaccaaggt gaagcggaag
     4201 aatctaaacc cgcactggaa tgagaccttt ctatttgaag ggtttcccta cgagaaagtg
     4261 gtgcagaggg tcctctacct ccaggtcctg gattatgacc gtttcagccg caatgacccc
     4321 attggggagg tgtccatccc tctgaacaag gtggacctga cccagatgca gaccttctgg
     4381 aaggatctga agccatgcag cgatgggagt gggagccgag gggagctgct cttgtccctc
     4441 tgctacaacc cctctgccaa ctccatcatc gtgaacatca tcaaagctcg aaacctcaaa
     4501 gccatggaca tcgggggcac atcagacccc tatgtgaagg tgtggctgat gtataaagac
     4561 aagcgggtag agaaaaagaa gactgtgaca aagaagagga acctgaaccc catcttcaat
     4621 gagtctttcg ccttcgacat acccacggag aagctgaggg agaccacgat catcatcact
     4681 gtcatggaca aagacaagct cagccgcaac gacgtcatcg gcaagatcta cctgtcctgg
     4741 aagagcggac caggtgaagt gaaacactgg aaggacatga tcgctcgtcc ccggcagcct
     4801 gtggcccagt ggcaccagct gaaagcctga aagcttaggt atttattcgg cgcaaagtgc
     4861 gtcgggtgat gctgccaact tagtcgagca ccaccaccac caccactgag atccggctgc
     4921 taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata
     4981 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc
     5041 cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     5101 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     5161 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     5221 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     5281 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     5341 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     5401 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     5461 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca
     5521 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     5581 tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat
     5641 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     5701 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     5761 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     5821 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct
     5881 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
     5941 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa
     6001 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     6061 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc
     6121 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     6181 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     6241 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
     6301 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     6361 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata
     6421 acaccccttg tattactgtt tatgtaagc
//
