LOCUS       sequence-383653-        5406 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmid encoding GST-tagged SYT9 C2B domain 
            with D330, 332N mutations.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5406)
  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 5406)
  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 C2B D330, 332N
FEATURES             Location/Qualifiers
     source          1..5406
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(75..80,81..96,97..103)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    111..127
                     /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             150..803
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     761..783
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             810..827
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(1348..1370)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1515..1533)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1601..1705
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1706..1774,1775..2566)
                     /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(1924..1943)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2737..3325
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3226..3245
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3479..3496
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3569..3646
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3647..4729
                     /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(3666..3685)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4778..4783,4784..4801,4802..4808)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4816..4832
                     /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     4840..4856
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4840..4856
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4852..5379
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4872..4889)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4872..4888)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4881..4903)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 gcgcactccc gttctggata atgttttttg cgccgacatc ataacggttc tggcaaatat
       61 tctgaaatga gctgttgaca attaatcatc ggctcgtata atgtgtggaa ttgtgagcgg
      121 ataacaattt cacacaggaa acagtattca tgtcccctat actaggttat tggaaaatta
      181 agggccttgt gcaacccact cgacttcttt tggaatatct tgaagaaaaa tatgaagagc
      241 atttgtatga gcgcgatgaa ggtgataaat ggcgaaacaa aaagtttgaa ttgggtttgg
      301 agtttcccaa tcttccttat tatattgatg gtgatgttaa attaacacag tctatggcca
      361 tcatacgtta tatagctgac aagcacaaca tgttgggtgg ttgtccaaaa gagcgtgcag
      421 agatttcaat gcttgaagga gcggttttgg atattagata cggtgtttcg agaattgcat
      481 atagtaaaga ctttgaaact ctcaaagttg attttcttag caagctacct gaaatgctga
      541 aaatgttcga agatcgttta tgtcataaaa catatttaaa tggtgatcat gtaacccatc
      601 ctgacttcat gttgtatgac gctcttgatg ttgttttata catggaccca atgtgcctgg
      661 atgcgttccc aaaattagtt tgttttaaaa aacgtattga agctatccca caaattgata
      721 agtacttgaa atccagcaag tatatagcat ggcctttgca gggctggcaa gccacgtttg
      781 gtggtggcga ccatcctcca aaatcggatc tggttccgcg tggatccaaa gaggagcaag
      841 agaaactggg ggacatctgc ttctctctcc gttatgtccc cacggccggg aagctcaccg
      901 tcattgtctt ggaagctaaa aacctgaaga aaatggatgt aggaggactc tcagatccct
      961 atgtgaaggt gcacctgctc cagggaggta aaaaggttcg caagaagaaa accaccatta
     1021 agaagaacac cctgaacccc tattacaacg aggccttcag ctttgaggtg ccctgtgacc
     1081 aggtgcagaa agtccaggtg gagctgactg ttctgaatta taacaaactg gggaagaatg
     1141 aggccatcgg gagagtggcg gtgggcgcag cagtcggtgg ggctggcctg cggcactggg
     1201 ctgacatgtt ggccaacccc aggcgcccca ttgcccagtg gcactctctg cggccccctg
     1261 acagagccag gccaatacct gcaccctgag aattcatcgt gactgactga cgatctgcct
     1321 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     1381 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     1441 tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtataattc
     1501 ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     1561 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     1621 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     1681 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     1741 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     1801 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     1861 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     1921 tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg
     1981 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     2041 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     2101 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     2161 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     2221 aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt
     2281 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     2341 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     2401 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     2461 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     2521 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     2581 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     2641 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     2701 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     2761 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     2821 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     2881 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     2941 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     3001 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     3061 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     3121 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     3181 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     3241 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     3301 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     3361 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     3421 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     3481 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     3541 gtgcggtatt tcacaccgca taaattccga caccatcgaa tggcgcaaaa cctttcgcgg
     3601 tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac
     3661 gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa
     3721 ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct
     3781 gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg
     3841 cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc
     3901 tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga
     3961 agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa
     4021 ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc
     4081 gttatttctt gatgtctctg accagacacc catcaacagt attattttct cccatgaaga
     4141 cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt
     4201 agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct
     4261 cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg
     4321 ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc
     4381 caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg
     4441 tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc
     4501 gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct
     4561 gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa
     4621 aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     4681 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     4741 ttaatgtaag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     4801 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
     4861 attacggatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     4921 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     4981 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg ctttgcctgg
     5041 tttccggcac cagaagcggt gccggaaagc tggctggagt gcgatcttcc tgaggccgat
     5101 actgtcgtcg tcccctcaaa ctggcagatg cacggttacg atgcgcccat ctacaccaac
     5161 gtgacctatc ccattacggt caatccgccg tttgttccca cggagaatcc gacgggttgt
     5221 tactcgctca catttaatgt tgatgaaagc tggctacagg aaggccagac gcgaattatt
     5281 tttgatggcg ttggaattag cttatcgact gcacggtgca ccaatgcttc tggcgtcagg
     5341 cagccatcgg aagctgtggt atggctgtgc aggtcgtaaa tcactgcata attcgtgtcg
     5401 ctcaag
//
