LOCUS       sequence-331743-        5809 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes cytoplasmic domain of Doc2beta for bacterial expression and 
            purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5809)
  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 5809)
  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) WT
FEATURES             Location/Qualifiers
     source          1..5809
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..24,25..31)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    39..55
                     /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     63..79
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     63..79
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             75..602
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(95..112)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(95..111)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(104..126)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(704..709,710..725,726..732)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    740..756
                     /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)."
     CDS             779..1432
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1390..1412
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             1439..1456
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(2380..2402)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2547..2565)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2633..2737
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2738..2806,2807..3598)
                     /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(2956..2975)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3769..4357
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4258..4277
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4511..4528
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4601..4678
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4679..5761
                     /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(4698..4717)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
       61 cacaggaaac agctatgacc atgattacgg attcactggc cgtcgtttta caacgtcgtg
      121 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
      181 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
      241 atggcgaatg gcgctttgcc tggtttccgg caccagaagc ggtgccggaa agctggctgg
      301 agtgcgatct tcctgaggcc gatactgtcg tcgtcccctc aaactggcag atgcacggtt
      361 acgatgcgcc catctacacc aacgtgacct atcccattac ggtcaatccg ccgtttgttc
      421 ccacggagaa tccgacgggt tgttactcgc tcacatttaa tgttgatgaa agctggctac
      481 aggaaggcca gacgcgaatt atttttgatg gcgttggaat tagcttatcg actgcacggt
      541 gcaccaatgc ttctggcgtc aggcagccat cggaagctgt ggtatggctg tgcaggtcgt
      601 aaatcactgc ataattcgtg tcgctcaagg cgcactcccg ttctggataa tgttttttgc
      661 gccgacatca taacggttct ggcaaatatt ctgaaatgag ctgttgacaa ttaatcatcg
      721 gctcgtataa tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagtattcat
      781 gtcccctata ctaggttatt ggaaaattaa gggccttgtg caacccactc gacttctttt
      841 ggaatatctt gaagaaaaat atgaagagca tttgtatgag cgcgatgaag gtgataaatg
      901 gcgaaacaaa aagtttgaat tgggtttgga gtttcccaat cttccttatt atattgatgg
      961 tgatgttaaa ttaacacagt ctatggccat catacgttat atagctgaca agcacaacat
     1021 gttgggtggt tgtccaaaag agcgtgcaga gatttcaatg cttgaaggag cggttttgga
     1081 tattagatac ggtgtttcga gaattgcata tagtaaagac tttgaaactc tcaaagttga
     1141 ttttcttagc aagctacctg aaatgctgaa aatgttcgaa gatcgtttat gtcataaaac
     1201 atatttaaat ggtgatcatg taacccatcc tgacttcatg ttgtatgacg ctcttgatgt
     1261 tgttttatac atggacccaa tgtgcctgga tgcgttccca aaattagttt gttttaaaaa
     1321 acgtattgaa gctatcccac aaattgataa gtacttgaaa tccagcaagt atatagcatg
     1381 gcctttgcag ggctggcaag ccacgtttgg tggtggcgac catcctccaa aatcggatct
     1441 ggttccgcgt ggatccggat ctggatctac cgccctgggt acactggact tcagtctgct
     1501 ctatgaccag gagaacaacg cactgcactg caccatcagc aaagccaagg gcctgaagcc
     1561 gatggatcac aatggactgg ctgatcccta cgtcaaacta cacttgctgc ctggagccag
     1621 caaggcaaat aagctcagaa caaaaaccct acggaacact ctgaacccct cctggaacga
     1681 gaccctcacc tattacggga tcacggatga ggacatgatc cgaaagaccc tgaggatctc
     1741 cgtgtgtgac gaggacaaat tccgccacaa cgagttcatc ggagagactc gagtgcccct
     1801 gaagaagctg aaacccaacc acaccaagac attcagcatc tgcctggaga agcagctgcc
     1861 ggtggacaag gcagaggaca agtccctgga ggagcgtggc cgcatcctca tctctctcaa
     1921 gtacagctca cagaagcagg gcctgctggt gggcatcgtt cgctgcgcac acctggcggc
     1981 catggatgct aacggctact cagaccccta cgtgaaaaca tatctgaaac cagatgtaga
     2041 caagaaatcc aagcataaga ccgcagtcaa gaagaaaaca ctaaaccctg aattcaatga
     2101 ggagttctgt tacgagatca agcatggaga cctagccaaa aagaccctgg aggtcactgt
     2161 ctgggactat gatattggaa aatccaatga tttcatcggt ggggtggttc tgggcatcaa
     2221 tgccaagggt gagcgcctga agcactggtt tgactgtctg aagaacaagg acaagaggat
     2281 tgagcgttgg cacacgctca ccaatgagct cccagggtag cggccgcatc gtgactgact
     2341 gacgatctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     2401 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     2461 gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg
     2521 agtgtataat tcttgaagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     2581 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     2641 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     2701 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     2761 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     2821 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     2881 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     2941 cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca
     3001 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     3061 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     3121 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     3181 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     3241 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt
     3301 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     3361 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     3421 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     3481 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     3541 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     3601 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     3661 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     3721 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     3781 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     3841 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     3901 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     3961 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     4021 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     4081 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4141 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     4201 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     4261 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     4321 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     4381 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     4441 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     4501 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     4561 ccttacgcat ctgtgcggta tttcacaccg cataaattcc gacaccatcg aatggcgcaa
     4621 aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg tggtgaatgt
     4681 gaaaccagta acgttatacg atgtcgcaga gtatgccggt gtctcttatc agaccgtttc
     4741 ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc
     4801 gatggcggag ctgaattaca ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc
     4861 gttgctgatt ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc
     4921 ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga tggtagaacg
     4981 aagcggcgtc gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg
     5041 gctgatcatt aactatccgc tggatgacca ggatgccatt gctgtggaag ctgcctgcac
     5101 taatgttccg gcgttatttc ttgatgtctc tgaccagaca cccatcaaca gtattatttt
     5161 ctcccatgaa gacggtacgc gactgggcgt ggagcatctg gtcgcattgg gtcaccagca
     5221 aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc tggctggctg
     5281 gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag gcgactggag
     5341 tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg ttcccactgc
     5401 gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta ccgagtccgg
     5461 gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag acagctcatg
     5521 ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt
     5581 ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt
     5641 ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct ctccccgcgc
     5701 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     5761 agcgcaacgc aattaatgta agttagctca ctcattaggc accccaggc
//
