LOCUS       sequence-317155-        5822 bp    DNA     circular SYN 15-APR-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 Apr 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) A298C
FEATURES             Location/Qualifiers
     source          1..5822
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..22,23..29)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..53
                     /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             76..729
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     687..709
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             736..753
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(1690..1712)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1857..1875)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1943..2047
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2048..2116,2117..2908)
                     /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(2266..2285)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3079..3667
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3568..3587
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3821..3838
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3911..3988
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3989..5071
                     /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(4008..4027)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5120..5125,5126..5143,5144..5150)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5158..5174
                     /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     5182..5198
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5182..5198
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5194..5721
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5214..5231)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5214..5230)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5223..5245)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag tattcatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa
      121 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc
      181 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt
      241 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata
      301 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt
      361 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt
      421 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat
      481 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg
      541 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa
      601 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc
      661 agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat
      721 cctccaaaat cggatctggt tccgcgtgga tccggatctg gatcagccct gggtacactg
      781 gacttcagtc tgctctatga ccaggagaac aacgcactgc acgcgaccat cagcaaagcc
      841 aagggcctga agccgatgga tcacaatgga ctggctgatc cctacgtcaa actacacttg
      901 ctgcctggag ccagcaaggc aaataagctc agaacaaaaa ccctacggaa cactctgaac
      961 ccctcctgga acgagaccct cacctattac gggatcacgg atgaggacat gatccgaaag
     1021 accctgagga tctccgtggc ggacgaggac aaattccgcc acaacgagtt catcggagag
     1081 actcgagtgc ccctgaagaa gctgaaaccc aaccacacca agacattcag catcgcgctg
     1141 gagaagcagc tgccggtgga caaggcagag gacaagtccc tggaggagcg tggccgcatc
     1201 ctcatctctc tcaagtacag ctcacagaag cagggcctgc tggtgggcat cgttcgcgcg
     1261 gcacacctgg cggccatgga ttgcaacggc tactcagacc cctacgtgaa aacatatctg
     1321 aaaccagatg tagacaagaa atccaagcat aagaccgcag tcaagaagaa aacactaaac
     1381 cctgaattca atgaggagtt cgcgtacgag atcaagcatg gagacctagc caaaaagacc
     1441 ctggaggtca ctgtctggga ctatgatatt ggaaaatcca atgatttcat cggtggggtg
     1501 gttctgggca tcaatgccaa gggtgagcgc ctgaagcact ggtttgacgc gctgaagaac
     1561 aaggacaaga ggattgagcg ttggcacacg ctcaccaatg agatcccagg ggctgtactc
     1621 agcgactgag cggccgcatc gtgactgact gacgatctgc ctcgcgcgtt tcggtgatga
     1681 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
     1741 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc
     1801 agccatgacc cagtcacgta gcgatagcgg agtgtataat tcttgaagac gaaagggcct
     1861 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     1921 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     1981 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     2041 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     2101 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     2161 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     2221 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     2281 attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     2341 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     2401 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     2461 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     2521 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     2581 cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     2641 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     2701 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     2761 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     2821 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     2881 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     2941 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     3001 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     3061 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     3121 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     3181 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     3241 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     3301 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     3361 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     3421 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     3481 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     3541 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     3601 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     3661 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     3721 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     3781 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     3841 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     3901 cataaattcc gacaccatcg aatggcgcaa aacctttcgc ggtatggcat gatagcgccc
     3961 ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga
     4021 gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc
     4081 tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg
     4141 cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct
     4201 ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg
     4261 tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt
     4321 gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca
     4381 ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc
     4441 tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt
     4501 ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc
     4561 tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca
     4621 gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca
     4681 aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct
     4741 gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt
     4801 gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca
     4861 ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca
     4921 ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc
     4981 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     5041 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca
     5101 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
     5161 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgga ttcactggcc
     5221 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     5281 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     5341 caacagttgc gcagcctgaa tggcgaatgg cgctttgcct ggtttccggc accagaagcg
     5401 gtgccggaaa gctggctgga gtgcgatctt cctgaggccg atactgtcgt cgtcccctca
     5461 aactggcaga tgcacggtta cgatgcgccc atctacacca acgtgaccta tcccattacg
     5521 gtcaatccgc cgtttgttcc cacggagaat ccgacgggtt gttactcgct cacatttaat
     5581 gttgatgaaa gctggctaca ggaaggccag acgcgaatta tttttgatgg cgttggaatt
     5641 agcttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
     5701 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
     5761 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
     5821 tg
//
