LOCUS       sequence-414436-        6828 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Octopus synaptotagmin-1 C2A domain with I248V mutation in 
            E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6828)
  AUTHORS   Birk MA, Liscovitch-Brauer N, Dominguez MJ, McNeme S, Yue Y, Hoff 
            JD, Twersky I, Verhey KJ, Sutton RB, Eisenberg E, Rosenthal JJC
  TITLE     Temperature-dependent RNA editing in octopus extensively recodes the
            neural proteome.
  JOURNAL   Cell. 2023 Jun 8;186(12):2544-2555.e13. doi: 
            10.1016/j.cell.2023.05.004. Epub 2023 Jun 8.
  PUBMED    37295402
REFERENCE   2  (bases 1 to 6828)
  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.cell.2023.05.004"; journalName: "Cell"; date: "2023-06-8-
            8"; volume: "186"; issue: "12"; pages: "2544-2555.e13"
COMMENT     Sequence Label: p202-Syt1-C2A-I248V
FEATURES             Location/Qualifiers
     source          1..6828
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     47..66
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        47..65
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    66..90
                     /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)."
     RBS             105..127
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             140..157
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             158..1252
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="IEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEK
                     FPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIA
                     YPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAAD
                     GGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETA
                     MTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLEN
                     YLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWY
                     AVRTVVINAASGRQTVDEALKDAQT"
     primer_bind     1226..1249
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1289..1300
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1304..1324
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1739..1756
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1809..1827)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1823..1870
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1907..2362
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1994..2013)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2204..2225
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2454..3269)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2454..3269)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3177..3196
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3261..3280
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3391..3979
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3880..3899
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4133..4150
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4165..4304
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4230..4249)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4390..4412
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4406..4597)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5406..6488)
                     /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     6450..6469
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6489..6566)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6772..6791
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact
       61 ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag
      121 aaggagatat accatggatc atcatcatca tcatcatatc gaagaaggta aactggtaat
      181 ctggattaac ggcgataaag gctataacgg tctcgctgaa gtcggtaaga aattcgagaa
      241 agataccgga attaaagtca ccgttgagca tccggataaa ctggaagaga aattcccaca
      301 ggttgcggca actggcgatg gccctgacat tatcttctgg gcacacgacc gctttggtgg
      361 ctacgctcaa tctggcctgt tggctgaaat caccccggac aaagcgttcc aggacaagct
      421 gtatccgttt acctgggatg ccgtacgtta caacggcaag ctgattgctt acccgatcgc
      481 tgttgaagcg ttatcgctga tttataacaa agatctgctg ccgaacccgc caaaaacctg
      541 ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa ggtaagagcg cgctgatgtt
      601 caacctgcaa gaaccgtact tcacctggcc gctgattgct gctgacgggg gttatgcgtt
      661 caagtatgaa aacggcaagt acgacattaa agacgtgggc gtggataacg ctggcgcgaa
      721 agcgggtctg accttcctgg ttgacctgat taaaaacaaa cacatgaatg cagacaccga
      781 ttactccatc gcagaagctg cctttaataa aggcgaaaca gcgatgacca tcaacggccc
      841 gtgggcatgg tccaacatcg acaccagcaa agtgaattat ggtgtaacgg tactgccgac
      901 cttcaagggt caaccatcca aaccgttcgt tggcgtgctg agcgcaggta ttaacgccgc
      961 cagtccgaac aaagagctgg caaaagagtt cctcgaaaac tatctgctga ctgatgaagg
     1021 tctggaagcg gttaataaag acaaaccgct gggtgccgta gcgctgaagt cttacgagga
     1081 agagttggcg aaagatccac gtattgccgc cacaatggaa aacgcccaga aaggtgaaat
     1141 catgccgaac atcccgcaga tgtccgcttt ctggtatgcc gtgcgtactg tggtgatcaa
     1201 cgccgccagc ggtcgtcaga ctgtcgatga agccctgaaa gacgcgcaga ctaattcgag
     1261 ctcagattat gatattccga ccaccgggat cgagggaagg ggagaaaatc tttattttca
     1321 aggtgatatt actcatatgg tgaagttggg gaaattgcaa tactctatgg attacgattt
     1381 ccaaaaagga gaacttaccg taaatgtcat ccaggcagcc gatttaccgg gcatggatat
     1441 gtctggaact tccgacccgt atgtcaaagt ttatctgatg cccgacaaaa agaaaaaatt
     1501 tgaaacgaaa gtacaccgta aaacgttgaa tccagtgttt aacgagagct ttactttcaa
     1561 gaatgtgcct tatgccgaca ttacgggaaa aacgctgatc ttcgctattt acgactttga
     1621 ccgtttttcc aagcacgatc aaattggcca agtccaggtt ccgatgaatt ccatcgatct
     1681 tggttctgtc gtcgaagaat ggcgcgattt aacgtcacca gacaactagt aactcgagca
     1741 ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc
     1801 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     1861 gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc
     1921 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     1981 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     2041 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     2101 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     2161 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     2221 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     2281 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     2341 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     2401 tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa
     2461 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat
     2521 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg
     2581 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat
     2641 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc
     2701 ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta
     2761 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
     2821 gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac
     2881 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct
     2941 aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga
     3001 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
     3061 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct
     3121 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg
     3181 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag
     3241 caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca
     3301 gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     3361 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     3421 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     3481 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     3541 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     3601 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     3661 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     3721 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     3781 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     3841 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     3901 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     3961 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     4021 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     4081 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     4141 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     4201 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     4261 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     4321 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     4381 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     4441 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     4501 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     4561 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     4621 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     4681 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     4741 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     4801 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     4861 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     4921 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     4981 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     5041 ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc
     5101 cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga
     5161 ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa
     5221 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa
     5281 gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     5341 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     5401 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     5461 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     5521 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     5581 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     5641 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg
     5701 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     5761 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
     5821 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
     5881 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
     5941 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
     6001 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
     6061 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
     6121 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
     6181 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
     6241 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
     6301 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
     6361 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
     6421 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
     6481 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     6541 aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg
     6601 cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg
     6661 tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac
     6721 gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc
     6781 ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggc
//
