LOCUS       sequence-383660-        5346 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Bacterial expression plasmid encoding GST-tagged SYT9 C2A domain 
            with D197, 199N mutations.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5346)
  AUTHORS   Seibert MJ, Evans CS, Stanley KS, Wu Z, Chapman ER
  TITLE     Synaptotagmin 9 Modulates Spontaneous Neurotransmitter Release in 
            Striatal Neurons by Regulating Substance P Secretion.
  JOURNAL   J Neurosci. 2023 Mar 1;43(9):1475-1491. doi: 
            10.1523/JNEUROSCI.1857-22.2023. Epub 2023 Feb 2.
  PUBMED    36732068
REFERENCE   2  (bases 1 to 5346)
  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: 
            "10.1523/JNEUROSCI.1857-22.2023"; journalName: "J Neurosci"; date: 
            "2023-03-1- 1"; volume: "43"; issue: "9"; pages: "1475-1491"
COMMENT     Sequence Label: pGEX SYT9 C2A D197,199N
FEATURES             Location/Qualifiers
     source          1..5346
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(71..76,77..92,93..99)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    107..123
                     /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             146..799
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     757..779
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             806..823
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(1284..1306)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1451..1469)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1537..1641
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1642..1710,1711..2502)
                     /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(1860..1879)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2673..3261
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3162..3181
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3415..3432
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3505..3582
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3583..4665
                     /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(3602..3621)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4714..4719,4720..4737,4738..4744)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4752..4768
                     /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     4776..4792
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4776..4792
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4788..5315
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4808..4825)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4808..4824)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4817..4839)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 actcccgttc tggataatgt tttttgcgcc gacatcataa cggttctggc aaatattctg
       61 aaatgagctg ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa
      121 caatttcaca caggaaacag tattcatgtc ccctatacta ggttattgga aaattaaggg
      181 ccttgtgcaa cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt
      241 gtatgagcgc gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt
      301 tcccaatctt ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat
      361 acgttatata gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat
      421 ttcaatgctt gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag
      481 taaagacttt gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat
      541 gttcgaagat cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga
      601 cttcatgttg tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc
      661 gttcccaaaa ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta
      721 cttgaaatcc agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg
      781 tggcgaccat cctccaaaat cggatctggt tccgcgtgga tccatgtcag acaaacacca
      841 gctaggccga ctgcagtatt cactggacta tgacttccag actggccagc tcctggtggg
      901 catcttgcaa gctcagggac tggctgcgtt ggacctagga ggttcctcag atccctatgt
      961 tagtgtctat ctgctgccag acaagcggag gcgacatgag accaaggtgc atcggcagac
     1021 cctgaatcca cactttggag agacatttgc cttcaaggtc ccatacgtgg aactaggggg
     1081 cagagtgctg gtcatggcgg tgtataactt taatcggttc tcccgcaacg atgccatcgg
     1141 ggaggtgcgg gtgcctatga gttcagtgaa cctggggcgg ccggtgcagg cctggagaga
     1201 gctgcaggtg gctcccaaag agtgagaatt catcgtgact gactgacgat ctgcctcgcg
     1261 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
     1321 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     1381 gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta taattcttga
     1441 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     1501 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     1561 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     1621 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     1681 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     1741 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     1801 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     1861 ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata
     1921 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     1981 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     2041 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     2101 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     2161 gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact
     2221 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     2281 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     2341 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     2401 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     2461 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     2521 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     2581 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     2641 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     2701 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     2761 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     2821 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     2881 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     2941 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     3001 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     3061 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     3121 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     3181 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     3241 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     3301 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     3361 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     3421 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     3481 ggtatttcac accgcataaa ttccgacacc atcgaatggc gcaaaacctt tcgcggtatg
     3541 gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc agtaacgtta
     3601 tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag
     3661 gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc ggagctgaat
     3721 tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct gattggcgtt
     3781 gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc
     3841 gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc
     3901 tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat cattaactat
     3961 ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt tccggcgtta
     4021 tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca tgaagacggt
     4081 acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg
     4141 ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa atatctcact
     4201 cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat gtccggtttt
     4261 caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct ggttgccaac
     4321 gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg
     4381 gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat cccgccgtta
     4441 accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa
     4501 ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga
     4561 aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     4621 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     4681 tgtaagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     4741 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
     4801 cggattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac
     4861 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca
     4921 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcttt gcctggtttc
     4981 cggcaccaga agcggtgccg gaaagctggc tggagtgcga tcttcctgag gccgatactg
     5041 tcgtcgtccc ctcaaactgg cagatgcacg gttacgatgc gcccatctac accaacgtga
     5101 cctatcccat tacggtcaat ccgccgtttg ttcccacgga gaatccgacg ggttgttact
     5161 cgctcacatt taatgttgat gaaagctggc tacaggaagg ccagacgcga attatttttg
     5221 atggcgttgg aattagctta tcgactgcac ggtgcaccaa tgcttctggc gtcaggcagc
     5281 catcggaagc tgtggtatgg ctgtgcaggt cgtaaatcac tgcataattc gtgtcgctca
     5341 aggcgc
//
