LOCUS       sequence-211257-        6515 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  IP2.0 for C. elegans (Codon optimization and introns)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6515)
  AUTHORS   Hara-Kuge S, Nishihara T, Matsuda T, Kitazono T, Teramoto T, Nagai 
            T, Ishihara T
  TITLE     An improved inverse-type Ca2+ indicator can detect putative neuronal
            inhibition in Caenorhabditis elegans by increasing signal intensity 
            upon Ca2+ decrease.
  JOURNAL   PLoS One. 2018 Apr 25;13(4):e0194707. doi: 
            10.1371/journal.pone.0194707. eCollection 2018.
  PUBMED    29694380
REFERENCE   2  (bases 1 to 6515)
  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"; journalName: "PLoS One.";
            date: "2018-04-25"; volume: "13(4):e0194707. doi"; pages: " 
            10.1371/journal.pone.0194707. eCollection 2018"
COMMENT     Sequence Label: pDEST- IP2.0(codon for C.elegans)
FEATURES             Location/Qualifiers
     source          1..6515
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    14..138
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(167..172,173..190,191..197)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             553..858
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     737..756
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     terminator      991..1077
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1169..1196
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     protein_bind    complement(1318..1442)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             1483..1500
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(1540..1548,1549..1563)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             1600..1677
                     /codon_start=1
                     /product="calmodulin-binding peptide"
                     /label=CBP
                     /note="derived from skeletal muscle myosin light chain 
                     kinase; binds calmodulin with nanomolar affinity in the 
                     presence of calcium"
                     /translation="KRRWKKNFIAVSAANRFKKISSSGAL"
     primer_bind     complement(3955..3977)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4077..4096
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4174..4192)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4260..4364
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4365..4433,4434..5225)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4583..4602)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5396..5984
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5885..5904
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6138..6155
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6272..6293
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6308..6313,6314..6331,6332..6338)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6346..6362
                     /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     6351..6373
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6370..6386
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6370..6386
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 aagctaccca tcaacaagtt tgtacaaaaa agctgaacga gaaacgtaaa atgatataaa
       61 tatcaatata ttaaattaga ttttgcataa aaaacagact acataatact gtaaaacaca
      121 acatatccag tcactatggc ggccggccgc attaggcacc ccaggcttta cactttatgc
      181 ttccggctcg tataatgtgt ggattttgag ttaggatcga tccggcttac taaaagccag
      241 ataacagtat gcgtatttgc gcgctgattt ttgcggtata agaatatata ctgatatgta
      301 tacccgaagt atgtcaaaaa gaggtatgct atgaagcagc gtattacagt gacagttgac
      361 agcgacagct atcagttgct caaggcatat atgatgtcaa tatctccggt ctggtaagca
      421 caaccatgca gaatgaagcc cgtcgtctgc gtgccgaacg ctggaaagcg gaaaatcagg
      481 aagggatggc tgaggtcgcc cggtttattg aaatgaacgg ctcttttgct gacgagaaca
      541 ggggctggtg aaatgcagtt taaggtttac acctataaaa gagagagccg ttatcgtctg
      601 tttgtggatg tacagagtga tattattgac acgcccgggc gacggatggt gatccccctg
      661 gccagtgcac gtctgctgtc agataaagtc tcccgtgaac tttacccggt ggtgcatatc
      721 ggggatgaaa gctggcgcat gatgaccacc gatatggcca gtgtgccggt ctccgttatc
      781 ggggaagaag tggctgatct cagccaccgc gaaaatgaca tcaaaaacgc cattaacctg
      841 atgttctggg gaatataaat gtcaggctcc cttatacaca gccagtctgc agggtcccac
      901 ctgaccccat gccgaactca gaagtgaaac gccgtagcgc cgatggtagt gtggggtctc
      961 cccatgcgag agtagggaac tgccaggcat caaataaaac gaaaggctca gtcgaaagac
     1021 tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg
     1081 ccgggagcgg atttgaacgt tgcgaagcaa cggcccggag ggtggcgggc aggacgcccg
     1141 ccataaactg ccaggcatca aattaagcag aaggccatcc tgacggatgg cctttttgcg
     1201 tttctacaaa ctcttcctgt cgtcatatct acaagccatc cccccacaga tacggtaaac
     1261 tagcctcgtt tttgcatcag gaaagcagct atgaaccact ccttaatgca ggtcgaccat
     1321 agtgactgga tatgttgtgt tttacagtat tatgtagtct gttttttatg caaaatctaa
     1381 tttaatatat tgatatttat atcattttac gtttctcgtt cagctttctt gtacaaagtg
     1441 gttgatcccg atatcttggt accggtagaa aaaatgggat ctcaccacca ccaccaccac
     1501 ggaatgacct ctatgaccgg aggacaacaa atgggacgcg acctttacga tgacgacgac
     1561 aaggaccttg ctaccgagtt catggttgat tcttctatga agcgccgttg gaagaagaac
     1621 ttcatcgctg tctccgccgc taaccgcttc aagaagattt cttcttctgg agcccttgga
     1681 tctgctggat acaactctac caacgtctac atcatggctg ataagcaaaa gaacggaatt
     1741 aaggctaact tcaagatccg tcataacatc gaggatggag gagttcaact tgctgatcat
     1801 taccaacaaa acactccaat cggagatgga ccagttcttc ttccagataa ccactacctt
     1861 tccttccaat ccgctctctc taaggtaagt ttaaacatat atatactaac taaccctgat
     1921 tatttaaatt ttcaggaccc aaacgagaag cgtgaccaca tggtccttct tgagttcgtc
     1981 accgctgctg gaatcaccct tggaatggat gagctttaca aggttgatgg aggatctgga
     2041 ggaactggag tttctaaggg agaggagctt ttcactggag ttgtcccaat ccttgtcgag
     2101 cttgatggag atgttaacgg acacaagttc tctgtttctg gagagggaga gggagacgct
     2161 acctacggaa agctcaccct caagctcatc tgcactaccg gaaagctccc agttccatgg
     2221 ccaacccttg ttaccaccct tggatacgga ctcaagtgtt tcgctcgtta cccagatcac
     2281 atgaagcaac atgatttctt caagtctgct atgccagagg gatacgttca agagcgtact
     2341 atcttcttca aggtaagttt aaacagttcg gtactaacta accatacata tttaaatttt
     2401 caggatgatg gaaactacaa gacccgtgct gaggttaagt tcgagggaga tacccttgtc
     2461 aaccgtatcg agcttaaggg aattgatttc aaggaagatg gaaacatcct tggacataag
     2521 ctcgaataca acggaactga tcaacttact gaggaacaaa tcgctgaatt caaggaggct
     2581 ttctcccttt tcgataagga cggagatgga actatcacta ccaaggtaag tttaaacatg
     2641 attttactaa ctaactaatc tgatttaaat tttcaggagc ttggaaccgt catgcgttct
     2701 ctcggacaaa acccaactga ggctgagctt caagacatga tcaacgaggt tgacgctgac
     2761 ggaaacggaa ctatttactt cccagagttc ctcaccatga tggcccgcaa gatgaaggat
     2821 accgattctg aggaggagat ccgcgaggct ttccgtgtct tcgataagga cggaaacgga
     2881 tacatttccg ccgcccaact tcgtcacgtt atgactaacc ttggagagaa gctcaccgac
     2941 gaggaggttg acgagatgat ccgtgaggct gacatcgacg gagacggaca agtcaactac
     3001 gaggagttcg ttcaaatgat gactgccaag taatctagac aattccaact gagcgccggt
     3061 cgctaccatt accaacttgt ctggtgtcaa aaataatagg ggccgctgtc atcagagtaa
     3121 gtttaaactg agttctacta actaacgagt aatatttaaa ttttcagcat ctcgcgcccg
     3181 tgcctctgac ttctaagtcc aattactctt caacatccct acatgctctt tctccctgtg
     3241 ctcccacccc ctatttttgt tattatcaaa aaacttctct taatttcttt gttttttagc
     3301 ttcttttaag tcacctctaa caatgaaatt gtgtagattc aaaaatagaa ttaattcgta
     3361 ataaaaagtc gaaaaaaatt gtgctccctc cccccattaa taataattct atcccaaaat
     3421 ctacacaatg ttctgtgtac acttcttatg ttttttactt ctgataaatt tttttgaaac
     3481 atcatagaaa aaaccgcaca caaaatacct tatcatatgt tacgtttcag tttatgaccg
     3541 caatttttat ttcttcgcac gtctgggcct ctcatgacgt caaatcatgc tcatcgtgaa
     3601 aaagttttgg agtatttttg gaatttttca atcaagtgaa agtttatgaa attaattttc
     3661 ctgcttttgc tttttggggt ttcccctatt gtttgtcaag atttcgagga cggcgttttt
     3721 cttgctaaaa tcacaagtat tgatgagcac gatgcaagaa agatcggaag aaggtttggg
     3781 tttgaggctc agtggaaggt gagtagaagt tgataatttg aaagtggagt agtgtctatg
     3841 gggtttttgc cttaaatgac agaatacatt cccaatatac caaacataac tgtttcctac
     3901 tagtcggccg tacgggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg
     3961 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     4021 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc
     4081 atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt
     4141 aaggagaaaa taccgcatca ggcggcctta agggcctcgt gatacgccta tttttatagg
     4201 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     4261 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     4321 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     4381 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     4441 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     4501 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     4561 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     4621 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     4681 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     4741 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     4801 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     4861 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     4921 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     4981 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     5041 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     5101 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     5161 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     5221 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     5281 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     5341 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     5401 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     5461 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     5521 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     5581 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     5641 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     5701 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     5761 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     5821 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     5881 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     5941 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     6001 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     6061 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     6121 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     6181 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     6241 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
     6301 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
     6361 aatttcacac aggaaacagc tatgaccatg attacgccaa gctgtaagtt taaacatgat
     6421 cttactaact aactattctc atttaaattt tcagagctta aaaatggctg aaatcactca
     6481 caacgatgga tacgctaaca acttggaaat gaaat
//
