LOCUS       sequence-337018-        5928 bp    DNA     circular SYN 15-APR-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 5928)
  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 5928)
  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: 
            "10.1016/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) I367T V304C
FEATURES             Location/Qualifiers
     source          1..5928
                     /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(1978..2000)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2145..2163)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2231..2335
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2336..2404,2405..3196)
                     /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(2554..2573)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3367..3955
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3856..3875
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4109..4126
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4199..4276
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4277..5359
                     /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(4296..4315)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5408..5413,5414..5431,5432..5438)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5446..5462
                     /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     5470..5486
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5470..5486
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5482..5928,1..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(5502..5519)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5502..5518)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5511..5533)
                     /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 gcttctccaa gcacgacatc atcggagagt
     1381 tcaaagttcc tatgaacacc gtggattttg gccatgtgac cgaggagtgg cgcgatctcc
     1441 agagcgctga gaaagaagag caagagaaac tgggtgacat cgccttctcc ctccgctacg
     1501 tccctactgc cggcaaactg actgttgtca ttctggaagc caagaacctg aagaagatgg
     1561 attgtggtgg cttatctgat ccctacgtga agattcacct gatgcagaac ggtaagaggc
     1621 tgaagaagaa aaagacgacg attaagaaga acacactcaa cccctactac aacgagtcct
     1681 tcagctttga agttccgttc gagcaaatcc agaaagtgca agtggtggta actgttttgg
     1741 actatgacaa gactggcaag aacgacgcca tcggaaaagt cttcgttggt tacaacagca
     1801 ctggggcgga gctgcgacac tggtcagaca tgctggccaa cccccggcga cccatcgcac
     1861 agtggcacac tctgcaggta gaggaggagg ttgatgccat gctggctgtc aagaagtaag
     1921 aattcatcgt gactgactga cgatctgcct cgcgcgtttc ggtgatgacg gtgaaaacct
     1981 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag
     2041 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca
     2101 gtcacgtagc gatagcggag tgtataattc ttgaagacga aagggcctcg tgatacgcct
     2161 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     2221 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     2281 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     2341 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     2401 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     2461 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     2521 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt
     2581 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     2641 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     2701 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     2761 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     2821 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc
     2881 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     2941 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     3001 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     3061 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     3121 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     3181 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     3241 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     3301 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3361 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3421 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3481 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     3541 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     3601 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3661 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3721 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3781 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3841 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     3901 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     3961 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     4021 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     4081 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     4141 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca taaattccga
     4201 caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca
     4261 attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt
     4321 ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg
     4381 ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca
     4441 actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc
     4501 gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt
     4561 ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct
     4621 cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc
     4681 tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc
     4741 catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt
     4801 cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg
     4861 tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga
     4921 acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga
     4981 gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg
     5041 cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga
     5101 taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct
     5161 gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg
     5221 caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatacgca
     5281 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     5341 actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac
     5401 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
     5461 aatttcacac aggaaacagc tatgaccatg attacggatt cactggccgt cgttttacaa
     5521 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     5581 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     5641 agcctgaatg gcgaatggcg ctttgcctgg tttccggcac cagaagcggt gccggaaagc
     5701 tggctggagt gcgatcttcc tgaggccgat actgtcgtcg tcccctcaaa ctggcagatg
     5761 cacggttacg atgcgcccat ctacaccaac gtgacctatc ccattacggt caatccgccg
     5821 tttgttccca cggagaatcc gacgggttgt tactcgctca catttaatgt tgatgaaagc
     5881 tggctacagg aaggccagac gcgaattatt tttgatggcg ttggaatt
//
