LOCUS       sequence-416670-        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     448..467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     701..718
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    733..872
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(798..817)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     958..980
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(974..1165)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1974..3056)
                     /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     3018..3037
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3057..3134)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3340..3359
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3443..3462
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3443..3461
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3462..3486
                     /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             3501..3523
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3543..3560
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3570..3587
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3594..3887
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             4813..4830
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4883..4901)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4897..4944
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4981..5436
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5068..5087)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5278..5299
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5528..6343)
                     /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(5528..6343)
                     /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     6251..6270
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6335..6354
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(6465..6506,1..547)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
       61 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
      121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      241 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      301 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      361 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      421 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      481 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      541 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      601 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
      661 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
      721 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
      781 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
      841 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
      901 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
      961 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     1021 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     1081 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     1141 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     1201 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     1261 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     1321 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     1381 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     1441 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     1501 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     1561 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     1621 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     1681 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     1741 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     1801 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     1861 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     1921 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     1981 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     2041 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     2101 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     2161 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     2221 ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac
     2281 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     2341 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     2401 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     2461 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     2521 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     2581 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     2641 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     2701 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     2761 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     2821 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     2881 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     2941 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     3001 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     3061 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     3121 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     3181 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     3241 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     3301 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     3361 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
     3421 cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac
     3481 aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacat atgggcagca
     3541 gccatcatca tcatcatcac ggcagcggcc tggtgccgcg cggcagcgct agcatgtcgg
     3601 actcagaagt caatcaagaa gctaagccag aggtcaagcc agaagtcaag cctgagactc
     3661 acatcaattt aaaggtgtcc gatggatctt cagagatctt cttcaagatc aaaaagacca
     3721 ctcctttaag aaggctgatg gaagcgttcg ctaaaagaca gggtaaggaa atggactcct
     3781 taagattctt gtacgacggt attagaattc aagctgatca gacccctgaa gatttggaca
     3841 tggaggataa cgatattatt gaggctcaca gagaacagat tggtggatcc gccctgggta
     3901 cactggactt cagtctgctc tatgaccagg agaacaacgc actgcacgcg accatcagca
     3961 aagccaaggg cctgaagccg atggatcaca atggactggc tgatccctac gtcaaactac
     4021 acttgctgcc tggagccagc aaggcaaata agctcagaac aaaaacccta cggaacactc
     4081 tgaacccctc ctggaacgag accctcacct attacgggat cacggatgag gacatgatcc
     4141 gaaagaccct gaggatctcc gtggcggacg aggacaaatt ccgccacaac gagttcatcg
     4201 gagagactcg agtgcccctg aagaagctga aacccaacca caccaagaca ttcagcatcg
     4261 cgctggagaa gcagctgccg gtggacaagg cagaggacaa gtccctggag gagcgtggcc
     4321 gcatcctcat ctctctcaag tacagctcac agaagcaggg cctgctggtg ggcatcgttc
     4381 gcgcggcaca cctggcggcc atggatgcta acggctactc agacccctac gtgaaaacat
     4441 atctgaaacc agatgtagac aagaaatcca agcataagac cgcagtcaag aagaaaacac
     4501 taaaccctga attcaatgag gagttcgcgt acgagatcaa gcatggagac ctagccaaaa
     4561 agaccctgga ggtcactgtc tgggactatg atatttgcaa atccaatgat ttcatcggtg
     4621 gggtggttct gggcatcaat gccaagggtg agcgcctgaa gcactggttt gacgcgctga
     4681 agaacaagga caagaggatt gagcgttggc acacgctcac caatgagatc ccaggggctg
     4741 tactcagcga ctgaaagctt aggtatttat tcggcgcaaa gtgcgtcggg tgatgctgcc
     4801 aacttagtcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag
     4861 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     4921 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
     4981 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     5041 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     5101 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     5161 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     5221 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     5281 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     5341 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     5401 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
     5461 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa
     5521 ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat
     5581 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc
     5641 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa
     5701 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag
     5761 tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa
     5821 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc
     5881 gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag
     5941 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat
     6001 caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc
     6061 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca
     6121 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt
     6181 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt
     6241 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta
     6301 atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac
     6361 tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg
     6421 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     6481 ctgcgcgtaa tctgctgctt gcaaac
//
