LOCUS       sequence-384177-        5799 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmid encoding GST-tagged SYT9 C2AB domain 
            with D197, 199, 330, 332N mutations.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5799)
  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 5799)
  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: 
            "10.1523/JNEUROSCI.1857-22.2023"; journalName: "J Neurosci"; date: 
            "2023-03-1- 1"; volume: "43"; issue: "9"; pages: "1475-1491"
COMMENT     Sequence Label: pGEX SYT9 C2AB D197,199,330,332N
FEATURES             Location/Qualifiers
     source          1..5799
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(173..178,179..194,195..201)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    209..225
                     /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             248..901
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     859..881
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             908..925
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(1839..1861)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2006..2024)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2092..2196
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2197..2265,2266..3057)
                     /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(2415..2434)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3228..3816
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3717..3736
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3970..3987
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4060..4137
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4138..5220
                     /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(4157..4176)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5269..5274,5275..5292,5293..5299)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5307..5323
                     /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     5331..5347
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5331..5347
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5343..5799,1..71)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5363..5380)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5363..5379)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5372..5394)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg gtatggctgt
       61 gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt tctggataat
      121 gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc tgttgacaat
      181 taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca cacaggaaac
      241 agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc aacccactcg
      301 acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc gcgatgaagg
      361 tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc ttccttatta
      421 tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata tagctgacaa
      481 gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc ttgaaggagc
      541 ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact ttgaaactct
      601 caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag atcgtttatg
      661 tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt tgtatgacgc
      721 tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa aattagtttg
      781 ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat ccagcaagta
      841 tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc atcctccaaa
      901 atcggatctg gttccgcgtg gatccatgtc agacaaacac cagctaggcc gactgcagta
      961 ttcactggac tatgacttcc agactggcca gctcctggtg ggcatcttgc aagctcaggg
     1021 actggctgcg ttggacctag gaggttcctc agatccctat gttagtgtct atctgctgcc
     1081 agacaagcgg aggcgacatg agaccaaggt gcatcggcag accctgaatc cacactttgg
     1141 agagacattt gccttcaagg tcccatacgt ggaactaggg ggcagagtgc tggtcatggc
     1201 ggtgtataac tttaatcggt tctcccgcaa cgatgccatc ggggaggtgc gggtgcctat
     1261 gagttcagtg aacctggggc ggccggtgca ggcctggaga gagctgcagg tggctcccaa
     1321 agaggagcaa gagaaactgg gggacatctg cttctctctc cgttatgtcc ccacggccgg
     1381 gaagctcacc gtcattgtct tggaagctaa aaacctgaag aaaatggatg taggaggact
     1441 ctcagatccc tatgtgaagg tgcacctgct ccagggaggt aaaaaggttc gcaagaagaa
     1501 aaccaccatt aagaagaaca ccctgaaccc ctattacaac gaggccttca gctttgaggt
     1561 gccctgtgac caggtgcaga aagtccaggt ggagctgact gttctgaatt ataacaaact
     1621 ggggaagaat gaggccatcg ggagagtggc ggtgggcgca gcagtcggtg gggctggcct
     1681 gcggcactgg gctgacatgt tggccaaccc caggcgcccc attgcccagt ggcactctct
     1741 gcggccccct gacagagcca ggccaatacc tgcaccctga gaattcatcg tgactgactg
     1801 acgatctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     1861 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     1921 tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga
     1981 gtgtataatt cttgaagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     2041 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     2101 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     2161 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     2221 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     2281 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     2341 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     2401 acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa
     2461 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     2521 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     2581 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     2641 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     2701 gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg
     2761 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     2821 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     2881 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     2941 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     3001 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     3061 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     3121 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     3181 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     3241 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     3301 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     3361 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     3421 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     3481 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     3541 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     3601 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     3661 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     3721 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     3781 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     3841 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     3901 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     3961 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     4021 cttacgcatc tgtgcggtat ttcacaccgc ataaattccg acaccatcga atggcgcaaa
     4081 acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg
     4141 aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc
     4201 cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg
     4261 atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg
     4321 ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg
     4381 gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga
     4441 agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg
     4501 ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact
     4561 aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc
     4621 tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa
     4681 atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg
     4741 cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt
     4801 gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg
     4861 atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg
     4921 ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt
     4981 tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg
     5041 gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc
     5101 tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg
     5161 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
     5221 gcgcaacgca attaatgtaa gttagctcac tcattaggca ccccaggctt tacactttat
     5281 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
     5341 ctatgaccat gattacggat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     5401 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     5461 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc
     5521 gctttgcctg gtttccggca ccagaagcgg tgccggaaag ctggctggag tgcgatcttc
     5581 ctgaggccga tactgtcgtc gtcccctcaa actggcagat gcacggttac gatgcgccca
     5641 tctacaccaa cgtgacctat cccattacgg tcaatccgcc gtttgttccc acggagaatc
     5701 cgacgggttg ttactcgctc acatttaatg ttgatgaaag ctggctacag gaaggccaga
     5761 cgcgaattat ttttgatggc gttggaatta gcttatcga
//
