LOCUS       sequence-336394-        5950 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of syt1 for purification in E. Coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5950)
  AUTHORS   Bradberry MM, Courtney NA, Dominguez MJ, Lofquist SM, Knox AT, 
            Sutton RB, Chapman ER
  TITLE     Molecular Basis for Synaptotagmin-1-Associated Neurodevelopmental 
            Disorder.
  JOURNAL   Neuron. 2020 Jul 8;107(1):52-64.e7. doi: 
            10.1016/j.neuron.2020.04.003. Epub 2020 May 1.
  PUBMED    32362337
REFERENCE   2  (bases 1 to 5950)
  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: 
            "10.1016/j.neuron.2020.04.003"; journalName: "Neuron"; date: 
            "2020-07-8- 8"; volume: "107"; issue: "1"; pages: "52-64.e7"
COMMENT     Sequence Label: pGEX Syt1 C2AB (96-421) D365E F234C
FEATURES             Location/Qualifiers
     source          1..5950
                     /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(2000..2022)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2167..2185)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2253..2357
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2358..2426,2427..3218)
                     /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(2576..2595)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3389..3977
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3878..3897
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4131..4148
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4221..4298
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4299..5381
                     /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(4318..4337)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5430..5435,5436..5453,5454..5460)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5468..5484
                     /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     5492..5508
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5492..5508
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5504..5950,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5524..5541)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5524..5540)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5533..5555)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 agcttatcga 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 gatccatggg aggaaagaac gccattaaca
      961 tgaaagacgt gaaagactta gggaagacca tgaaggatca ggcccttaag gatgacgatg
     1021 ctgaaaccgg actgactgat ggagaagaaa aggaagagcc caaggaagag gagaaactgg
     1081 gaaagctcca atattcactg gactatgact tccagaataa ccagctgttg gtgggaatca
     1141 tccaggctgc tgaactgccc gccctggaca tggggggtac atccgatcca tacgtcaaag
     1201 tcttcctgct gcctgacaaa aagaagaaat ttgagactaa agtccaccgg aaaaccctca
     1261 atccagtctt caatgaacaa tttactttca aggtacccta ctcggaatta ggtggcaaaa
     1321 ccctggtgat ggctgtgtat gactttgatc gctgttccaa gcacgacatc atcggagagt
     1381 tcaaagttcc tatgaacacc gtggattttg gccatgtgac cgaggagtgg cgcgatctcc
     1441 agagcgctga gaaagaagag caagagaaac tgggtgacat cgccttctcc ctccgctacg
     1501 tccctactgc cggcaaactg actgttgtca ttctggaagc caagaacctg aagaagatgg
     1561 atgtgggtgg cttatctgat ccctacgtga agattcacct gatgcagaac ggtaagaggc
     1621 tgaagaagaa aaagacgacg attaagaaga acacactcaa cccctactac aacgagtcct
     1681 tcagctttga agttccgttc gagcaaatcc agaaagtgca agtggtggta actgttttgg
     1741 actatgagaa gattggcaag aacgacgcca tcggaaaagt cttcgttggt tacaacagca
     1801 ctggggcgga gctgcgacac tggtcagaca tgctggccaa cccccggcga cccatcgcac
     1861 agtggcacac tctgcaggta gaggaggagg ttgatgccat gctggctgtc aagaagtaag
     1921 aattcccggg tcgactcgag cggccgcatc gtgactgact gacgatctgc ctcgcgcgtt
     1981 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     2041 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     2101 gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtataat tcttgaagac
     2161 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     2221 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     2281 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     2341 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     2401 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     2461 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     2521 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     2581 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact
     2641 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     2701 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     2761 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     2821 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     2881 agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     2941 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     3001 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     3061 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     3121 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     3181 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     3241 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     3301 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     3361 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     3421 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     3481 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     3541 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     3601 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     3661 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     3721 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     3781 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     3841 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     3901 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     3961 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     4021 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     4081 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     4141 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     4201 tttcacaccg cataaattcc gacaccatcg aatggcgcaa aacctttcgc ggtatggcat
     4261 gatagcgccc ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg
     4321 atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca
     4381 gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca
     4441 ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca
     4501 cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg
     4561 atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta
     4621 aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc
     4681 tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc
     4741 ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc
     4801 gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc
     4861 cattaagttc tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca
     4921 atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac
     4981 aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc
     5041 agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata
     5101 tctcggtagt gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca
     5161 ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct
     5221 ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa
     5281 ccaccctggc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     5341 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta
     5401 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     5461 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgga
     5521 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     5581 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     5641 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgctttgcct ggtttccggc
     5701 accagaagcg gtgccggaaa gctggctgga gtgcgatctt cctgaggccg atactgtcgt
     5761 cgtcccctca aactggcaga tgcacggtta cgatgcgccc atctacacca acgtgaccta
     5821 tcccattacg gtcaatccgc cgtttgttcc cacggagaat ccgacgggtt gttactcgct
     5881 cacatttaat gttgatgaaa gctggctaca ggaaggccag acgcgaatta tttttgatgg
     5941 cgttggaatt
//
