LOCUS       sequence-258879-        5815 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes cytoplasmic domain of synaptotagmin-1 for bacterial 
            expression, purification, and labeling.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5815)
  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 5815)
  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 Syt1 C2AB(140) V304C
FEATURES             Location/Qualifiers
     source          1..5815
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..211)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    219..235
                     /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             258..911
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     869..891
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             918..935
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(1865..1887)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2032..2050)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2118..2222
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2223..2291,2292..3083)
                     /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(2441..2460)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3254..3842
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3743..3762
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3996..4013
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4086..4163
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             4164..5246
                     /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(4183..4202)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5259..5280
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5295..5300,5301..5318,5319..5325)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5333..5349
                     /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     5357..5373
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5357..5373
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5369..5815,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGITLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5389..5406)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5389..5405)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5398..5420)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 acgttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatctg gttccgcgtg gatccggatc tggatctgag aaactgggaa
      961 agctccaata ttcactggac tatgacttcc agaataacca gctgttggtg ggaatcatcc
     1021 aggctgctga actgcccgcc ctggacatgg ggggtacatc cgatccatac gtcaaagtct
     1081 tcctgctgcc tgacaaaaag aagaaatttg agactaaagt ccaccggaaa accctcaatc
     1141 cagtcttcaa tgaacaattt actttcaagg taccctactc ggaattaggt ggcaaaaccc
     1201 tggtgatggc tgtgtatgac tttgatcgct tctccaagca cgacatcatc ggagagttca
     1261 aagttcctat gaacaccgtg gattttggcc atgtgaccga ggagtggcgt gatctccaga
     1321 gcgctgagaa agaagagcaa gagaaactgg gtgacatcgc cttctccctc cgctacgtcc
     1381 ctactgccgg caaactgact gttgtcattc tggaagccaa gaacctgaag aagatggatt
     1441 gtggtggctt atctgatccc tacgtgaaga ttcacctgat gcagaacggt aagaggctga
     1501 agaagaaaaa gacgacgatt aagaagaaca cactcaaccc ctactacaac gagtccttca
     1561 gctttgaagt tccgttcgag caaatccaga aagtgcaagt ggtggtaact gttttggact
     1621 atgacaagat tggcaagaac gacgccatcg gcaaagtctt cgttggttac aacagcactg
     1681 gggcggagct gcgacactgg tcagacatgc tggccaaccc ccggcgaccc atcgcacagt
     1741 ggcacactct gcaggtagag gaggaggttg atgccatgct ggctgtcaag aagtaagaat
     1801 tcatcgggcc gcatcgtgac tgactgacga tctgcctcgc gcgtttcggt gatgacggtg
     1861 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
     1921 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca
     1981 tgacccagtc acgtagcgat agcggagtgt ataattcttg aagacgaaag ggcctcgtga
     2041 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     2101 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     2161 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     2221 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     2281 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     2341 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     2401 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     2461 cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     2521 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     2581 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     2641 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     2701 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     2761 tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     2821 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     2881 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     2941 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     3001 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     3061 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     3121 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     3181 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     3241 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     3301 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     3361 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     3421 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     3481 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     3541 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     3601 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     3661 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     3721 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     3781 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     3841 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     3901 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     3961 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     4021 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcataa
     4081 attccgacac catcgaatgg tgcaaaacct ttcgcggtat ggcatgatag cgcccggaag
     4141 agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg
     4201 ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga
     4261 aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg
     4321 cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc
     4381 tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca
     4441 gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca
     4501 atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg
     4561 ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc
     4621 agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc
     4681 atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct
     4741 cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga
     4801 tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc
     4861 tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg
     4921 caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat
     4981 acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt
     5041 ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg
     5101 tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca
     5161 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     5221 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
     5281 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
     5341 ggataacaat ttcacacagg aaacagctat gaccatgatt acggattcac tggccgtcgt
     5401 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     5461 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     5521 gttgcgcagc ctgaatggcg aatggcgctt tgcctggttt ccggcaccag aagcggtgcc
     5581 ggaaagctgg ctggagtgcg atcttcctga ggccgatact gtcgtcgtcc cctcaaactg
     5641 gcagatgcac ggttacgatg cgcccatcta caccaacgta acctatccca ttacggtcaa
     5701 tccgccgttt gttcccacgg agaatccgac gggttgttac tcgctcacat ttaatgttga
     5761 tgaaagctgg ctacaggaag gccagacgcg aattattttt gatggcgttg gaatt
//
