LOCUS       sequence-317156-        5822 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes cytoplasmic domain of Doc2beta for bacterial expression, 
            purification, and labeling.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5822)
  AUTHORS   Bradberry MM, Bao H, Lou X, Chapman ER
  TITLE     Phosphatidylinositol 4,5-bisphosphate drives Ca(2+)-independent 
            membrane penetration by the tandem C2 domain proteins 
            synaptotagmin-1 and Doc2beta.
  JOURNAL   J Biol Chem. 2019 Jul 12;294(28):10942-10953. doi: 
            10.1074/jbc.RA119.007929. Epub 2019 May 30.
  PUBMED    31147445
REFERENCE   2  (bases 1 to 5822)
  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"; doi: 
            "10.1074/jbc.RA119.007929"; journalName: "J Biol Chem"; date: 
            "2019-07-12- 12"; volume: "294"; issue: "28"; pages: "10942-10953"
COMMENT     Sequence Label: pGEX-4T Doc2beta C2AB(125) G361C
FEATURES             Location/Qualifiers
     source          1..5822
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..654
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     612..634
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             661..678
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(1615..1637)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1782..1800)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1868..1972
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1973..2041,2042..2833)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2191..2210)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3004..3592
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3493..3512
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3746..3763
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3836..3913
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3914..4996
                     /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     complement(3933..3952)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5045..5050,5051..5068,5069..5075)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5083..5099
                     /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)."
     primer_bind     5107..5123
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5107..5123
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5119..5646
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5139..5156)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5139..5155)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5148..5170)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(5748..5753,5754..5769,5770..5776)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    5784..5800
                     /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)."
ORIGIN
        1 atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt
       61 ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa
      121 tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat
      181 ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac
      241 atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg
      301 gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt
      361 gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa
      421 acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat
      481 gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa
      541 aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca
      601 tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat
      661 ctggttccgc gtggatccgg atctggatca gccctgggta cactggactt cagtctgctc
      721 tatgaccagg agaacaacgc actgcacgcg accatcagca aagccaaggg cctgaagccg
      781 atggatcaca atggactggc tgatccctac gtcaaactac acttgctgcc tggagccagc
      841 aaggcaaata agctcagaac aaaaacccta cggaacactc tgaacccctc ctggaacgag
      901 accctcacct attacgggat cacggatgag gacatgatcc gaaagaccct gaggatctcc
      961 gtggcggacg aggacaaatt ccgccacaac gagttcatcg gagagactcg agtgcccctg
     1021 aagaagctga aacccaacca caccaagaca ttcagcatcg cgctggagaa gcagctgccg
     1081 gtggacaagg cagaggacaa gtccctggag gagcgtggcc gcatcctcat ctctctcaag
     1141 tacagctcac agaagcaggg cctgctggtg ggcatcgttc gcgcggcaca cctggcggcc
     1201 atggatgcta acggctactc agacccctac gtgaaaacat atctgaaacc agatgtagac
     1261 aagaaatcca agcataagac cgcagtcaag aagaaaacac taaaccctga attcaatgag
     1321 gagttcgcgt acgagatcaa gcatggagac ctagccaaaa agaccctgga ggtcactgtc
     1381 tgggactatg atatttgcaa atccaatgat ttcatcggtg gggtggttct gggcatcaat
     1441 gccaagggtg agcgcctgaa gcactggttt gacgcgctga agaacaagga caagaggatt
     1501 gagcgttggc acacgctcac caatgagatc ccaggggctg tactcagcga ctgagcggcc
     1561 gcatcgtgac tgactgacga tctgcctcgc gcgtttcggt gatgacggtg aaaacctctg
     1621 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     1681 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc
     1741 acgtagcgat agcggagtgt ataattcttg aagacgaaag ggcctcgtga tacgcctatt
     1801 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     1861 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     1921 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     1981 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     2041 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     2101 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     2161 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga
     2221 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     2281 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     2341 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     2401 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     2461 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc
     2521 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     2581 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     2641 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     2701 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     2761 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     2821 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     2881 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     2941 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     3001 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     3061 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     3121 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     3181 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     3241 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     3301 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     3361 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     3421 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     3481 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     3541 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     3601 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     3661 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     3721 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct
     3781 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcataa attccgacac
     3841 catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt
     3901 cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc
     3961 ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga
     4021 aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact
     4081 ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc
     4141 gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt
     4201 gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc
     4261 gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt
     4321 ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat
     4381 caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc
     4441 attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct
     4501 gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg
     4561 ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg
     4621 catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc
     4681 cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat acgacgatac
     4741 cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct
     4801 ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa
     4861 tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac
     4921 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     4981 ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta gctcactcat taggcacccc
     5041 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
     5101 ttcacacagg aaacagctat gaccatgatt acggattcac tggccgtcgt tttacaacgt
     5161 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     5221 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     5281 ctgaatggcg aatggcgctt tgcctggttt ccggcaccag aagcggtgcc ggaaagctgg
     5341 ctggagtgcg atcttcctga ggccgatact gtcgtcgtcc cctcaaactg gcagatgcac
     5401 ggttacgatg cgcccatcta caccaacgtg acctatccca ttacggtcaa tccgccgttt
     5461 gttcccacgg agaatccgac gggttgttac tcgctcacat ttaatgttga tgaaagctgg
     5521 ctacaggaag gccagacgcg aattattttt gatggcgttg gaattagctt atcgactgca
     5581 cggtgcacca atgcttctgg cgtcaggcag ccatcggaag ctgtggtatg gctgtgcagg
     5641 tcgtaaatca ctgcataatt cgtgtcgctc aaggcgcact cccgttctgg ataatgtttt
     5701 ttgcgccgac atcataacgg ttctggcaaa tattctgaaa tgagctgttg acaattaatc
     5761 atcggctcgt ataatgtgtg gaattgtgag cggataacaa tttcacacag gaaacagtat
     5821 tc
//
