LOCUS       sequence-421654-        6020 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of SUMO-tagged human cPLA2 C2 domain (Y96C).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6020)
  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 6020)
  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-cPLA2 C2 domain (Y96C)
FEATURES             Location/Qualifiers
     source          1..6020
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      54..642
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     543..562
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     796..813
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    828..967
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(893..912)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1053..1075
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1069..1260)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2069..3151)
                     /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     3113..3132
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3152..3229)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3435..3454
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3538..3557
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3538..3556
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3557..3581
                     /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             3596..3618
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3638..3655
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3665..3682
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3689..3982
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             4422..4439
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4492..4510)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4506..4553
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4590..5045
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4677..4696)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4887..4908
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5137..5952)
                     /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(5137..5952)
                     /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     5860..5879
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5944..5963
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
       61 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
      121 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
      181 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
      241 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
      301 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
      361 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
      421 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
      481 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
      541 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
      601 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
      661 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
      721 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
      781 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
      841 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
      901 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
      961 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     1021 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     1081 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     1141 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     1201 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     1261 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     1321 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     1381 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     1441 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     1501 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     1561 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     1621 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     1681 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc
     1741 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     1801 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     1861 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     1921 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     1981 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     2041 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     2101 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     2161 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     2221 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     2281 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     2341 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     2401 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     2461 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     2521 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     2581 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     2641 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     2701 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     2761 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     2821 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     2881 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     2941 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     3001 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     3061 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     3121 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     3181 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     3241 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     3301 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     3361 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     3421 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     3481 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa
     3541 tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt
     3601 ttaactttaa gaaggagata tacatatggg cagcagccat catcatcatc atcacggcag
     3661 cggcctggtg ccgcgcggca gcgctagcat gtcggactca gaagtcaatc aagaagctaa
     3721 gccagaggtc aagccagaag tcaagcctga gactcacatc aatttaaagg tgtccgatgg
     3781 atcttcagag atcttcttca agatcaaaaa gaccactcct ttaagaaggc tgatggaagc
     3841 gttcgctaaa agacagggta aggaaatgga ctccttaaga ttcttgtacg acggtattag
     3901 aattcaagct gatcagaccc ctgaagattt ggacatggag gataacgata ttattgaggc
     3961 tcacagagaa cagattggtg gatcctccca caagtttacg gtagtggtgt tacgtgccac
     4021 caaagtgaca aagggggcct ttggtgacat gcttgatact ccagatccct atgtggaact
     4081 ttttatctct acaacccctg acagcaggaa gagaacaaga catttcaata atgacataaa
     4141 ccctgtgtgg aatgagacct ttgaatttat tttggatcct aatcaggaaa atgttttgga
     4201 gattacgtta atggatgcca attgcgtcat ggatgaaact ctagggacag caacatttac
     4261 tgtatcttct atgaaggtgg gagaaaagaa agaagttcct tttattttca accaagtcac
     4321 tgaaatggtt ctagaaatgt ctcttgaagt tgcctcaagc tgaaagctta ggtatttatt
     4381 cggcgcaaag tgcgtcgggt gatgctgcca acttagtcga gcaccaccac caccaccact
     4441 gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc
     4501 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     4561 gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     4621 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     4681 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     4741 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     4801 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     4861 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     4921 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     4981 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     5041 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     5101 atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa
     5161 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt
     5221 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg
     5281 gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat
     5341 aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag
     5401 tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc
     5461 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg
     5521 atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc
     5581 cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt
     5641 tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt
     5701 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac
     5761 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc
     5821 atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc
     5881 atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg
     5941 aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca
     6001 tgaccaaaat cccttaacgt
//
