LOCUS       sequence-421655-        6446 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of SUMO-tagged mouse syt7 C2A-2A 2B.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6446)
  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 6446)
  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 C2A-2A 2B
FEATURES             Location/Qualifiers
     source          1..6446
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      10..598
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     499..518
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     752..769
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    784..923
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(849..868)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1009..1031
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1025..1216)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2025..3107)
                     /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     3069..3088
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3108..3185)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3391..3410
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3494..3513
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3494..3512
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3513..3537
                     /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             3552..3574
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3594..3611
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3621..3638
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3645..3938
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             4804..4821
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4874..4892)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4888..4935
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4972..5427
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5059..5078)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5269..5290
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5519..6334)
                     /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(5519..6334)
                     /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     6242..6261
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6326..6345
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
       61 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
      121 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
      181 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
      241 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
      301 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
      361 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
      421 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
      481 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
      541 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
      601 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
      661 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
      721 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      781 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc
      841 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
      901 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
      961 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     1021 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
     1081 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
     1141 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
     1201 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
     1261 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
     1321 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
     1381 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
     1441 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
     1501 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
     1561 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
     1621 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
     1681 gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac
     1741 cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga
     1801 tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca
     1861 cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc
     1921 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     1981 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     2041 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     2101 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     2161 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     2221 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     2281 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
     2341 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     2401 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     2461 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     2521 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     2581 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     2641 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     2701 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     2761 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     2821 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     2881 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     2941 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     3001 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     3061 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     3121 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     3181 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     3241 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
     3301 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
     3361 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
     3421 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc
     3481 gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa caattcccct
     3541 ctagaaataa ttttgtttaa ctttaagaag gagatataca tatgggcagc agccatcatc
     3601 atcatcatca cggcagcggc ctggtgccgc gcggcagcgc tagcatgtcg gactcagaag
     3661 tcaatcaaga agctaagcca gaggtcaagc cagaagtcaa gcctgagact cacatcaatt
     3721 taaaggtgtc cgatggatct tcagagatct tcttcaagat caaaaagacc actcctttaa
     3781 gaaggctgat ggaagcgttc gctaaaagac agggtaagga aatggactcc ttaagattct
     3841 tgtacgacgg tattagaatt caagctgatc agacccctga agatttggac atggaggata
     3901 acgatattat tgaggctcac agagaacaga ttggtggatc cggccgaatc cagttcagtg
     3961 ttggctacaa cttccaagag tccacactca ccgtgaaggt catgaaggcc caagagctgc
     4021 cagccaagga cgccagcggt actagtgacc cctttgtcaa gatctacctg ctacccgaca
     4081 agaagcacaa actggagacc aaggtgaagc ggaagaatct aaacccgcac tggaatgaga
     4141 cctttctatt tgaagggttt ccctacgaga aagtggtgca gagggtcctc tacctccagg
     4201 tcctggatta tgaccgtgca agccgcaatg accccattgg ggaggtgtcc atccctctga
     4261 acaaggtgga cctgacccag atgcagacct tctggaagga tctgaagcca tgcagcgatg
     4321 ggagtgggag ccgaggggag ctgctcttgt ccctctgcta caacccctct gccaactcca
     4381 tcatcgtgaa catcatcaaa gctcgaaacc tcaaagccat ggacatcggg ggcacatcag
     4441 acccctatgt gaaggtgtgg ctgatgtata aagacaagcg ggtagagaaa aagaagaccg
     4501 tgacaaagaa gaggaacctg aaccccatct tcaatgagtc tttcgccttc gacataccca
     4561 cggagaagct gagggagacc acgatcatca tcactgtcat ggacaaagac aagctcagcc
     4621 acaacgacgt catcggcaag atctacctgt cctggaagag cggaccaggt gaagtgaaac
     4681 actggaagga catgatcgct cgtccccggc agcctgtggc ccagtggcac cagctgaaag
     4741 cctgaaagct taggtattta ttcggcgcaa agtgcgtcgg gtgatgctgc caacttagtc
     4801 gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag
     4861 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
     4921 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
     4981 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     5041 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     5101 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     5161 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     5221 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     5281 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     5341 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     5401 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
     5461 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt
     5521 agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac
     5581 catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata
     5641 ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta
     5701 ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg
     5761 aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc
     5821 cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg
     5881 cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat
     5941 gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt
     6001 cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat
     6061 caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta
     6121 gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca
     6181 actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat
     6241 tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc
     6301 tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt
     6361 aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc gttccactga
     6421 gcgtcagacc ccgtagaaaa gatcaa
//
