LOCUS       sequence-421649-        6506 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of SUMO-tagged rat Doc2-beta C2AB (G361C).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6506)
  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 6506)
  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-Doc2-beta C2AB (G361C)
FEATURES             Location/Qualifiers
     source          1..6506
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     472..491
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     725..742
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    757..896
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(822..841)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     982..1004
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(998..1189)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1998..3080)
                     /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     3042..3061
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3081..3158)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3364..3383
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3467..3486
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3467..3485
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3486..3510
                     /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             3525..3547
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3567..3584
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3594..3611
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3618..3911
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             4837..4854
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4907..4925)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4921..4968
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5005..5460
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5092..5111)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5302..5323
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5552..6367)
                     /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(5552..6367)
                     /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     6275..6294
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6359..6378
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(6489..6506,1..571)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
       61 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
      121 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
      181 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
      241 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
      301 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
      361 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
      421 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
      481 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
      541 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
      601 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
      661 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
      721 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
      781 gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc
      841 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
      901 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
      961 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     1021 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     1081 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     1141 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     1201 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     1261 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     1321 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     1381 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     1441 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     1501 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     1561 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     1621 tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg
     1681 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt
     1741 gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt
     1801 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga
     1861 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg
     1921 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     1981 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     2041 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     2101 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     2161 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     2221 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata
     2281 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     2341 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     2401 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     2461 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     2521 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     2581 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     2641 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     2701 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     2761 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     2821 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     2881 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     2941 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     3001 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     3061 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     3121 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
     3181 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
     3241 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
     3301 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
     3361 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
     3421 cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact
     3481 ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag
     3541 aaggagatat acatatgggc agcagccatc atcatcatca tcacggcagc ggcctggtgc
     3601 cgcgcggcag cgctagcatg tcggactcag aagtcaatca agaagctaag ccagaggtca
     3661 agccagaagt caagcctgag actcacatca atttaaaggt gtccgatgga tcttcagaga
     3721 tcttcttcaa gatcaaaaag accactcctt taagaaggct gatggaagcg ttcgctaaaa
     3781 gacagggtaa ggaaatggac tccttaagat tcttgtacga cggtattaga attcaagctg
     3841 atcagacccc tgaagatttg gacatggagg ataacgatat tattgaggct cacagagaac
     3901 agattggtgg atccgccctg ggtacactgg acttcagtct gctctatgac caggagaaca
     3961 acgcactgca cgcgaccatc agcaaagcca agggcctgaa gccgatggat cacaatggac
     4021 tggctgatcc ctacgtcaaa ctacacttgc tgcctggagc cagcaaggca aataagctca
     4081 gaacaaaaac cctacggaac actctgaacc cctcctggaa cgagaccctc acctattacg
     4141 ggatcacgga tgaggacatg atccgaaaga ccctgaggat ctccgtggcg gacgaggaca
     4201 aattccgcca caacgagttc atcggagaga ctcgagtgcc cctgaagaag ctgaaaccca
     4261 accacaccaa gacattcagc atcgcgctgg agaagcagct gccggtggac aaggcagagg
     4321 acaagtccct ggaggagcgt ggccgcatcc tcatctctct caagtacagc tcacagaagc
     4381 agggcctgct ggtgggcatc gttcgcgcgg cacacctggc ggccatggat gctaacggct
     4441 actcagaccc ctacgtgaaa acatatctga aaccagatgt agacaagaaa tccaagcata
     4501 agaccgcagt caagaagaaa acactaaacc ctgaattcaa tgaggagttc gcgtacgaga
     4561 tcaagcatgg agacctagcc aaaaagaccc tggaggtcac tgtctgggac tatgatattt
     4621 gcaaatccaa tgatttcatc ggtggggtgg ttctgggcat caatgccaag ggtgagcgcc
     4681 tgaagcactg gtttgacgcg ctgaagaaca aggacaagag gattgagcgt tggcacacgc
     4741 tcaccaatga gatcccaggg gctgtactca gcgactgaaa gcttaggtat ttattcggcg
     4801 caaagtgcgt cgggtgatgc tgccaactta gtcgagcacc accaccacca ccactgagat
     4861 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     4921 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     4981 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     5041 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     5101 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     5161 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     5221 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     5281 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     5341 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     5401 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     5461 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     5521 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     5581 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     5641 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     5701 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     5761 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     5821 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     5881 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     5941 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     6001 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     6061 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     6121 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     6181 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     6241 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     6301 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat
     6361 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc
     6421 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     6481 ggatcttctt gagatccttt ttttct
//
