LOCUS       sequence-205941-        5544 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express IP2.0 in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5544)
  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 5544)
  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: pTorPE-IP2.0
FEATURES             Location/Qualifiers
     source          1..5544
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..285
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     208..227
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     RBS             327..338
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             493..510
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             514..546
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MTSMTGGQQMG"
     CDS             join(550..558,559..573)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             610..687
                     /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     918..939
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1051..1072)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1312..1331)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(1945..1962)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2115..2201
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2293..2320
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2339..2430
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2431..2499,2500..3291)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2649..2668)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3462..4050
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3951..3970
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4204..4221
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4236..4376
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4302..4321)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4462..4484
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4640..5518)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct
       61 tctcgctaac caaaccggta accccgctta ttaaaagcat tctgtaacaa agcgggacca
      121 aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg
      181 attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg
      241 atcctacctg acgcttttta tcgcaactct ctactgtttc tccatacccg ttttttgggc
      301 taacaggagg aattaaccat gggtttaaag aggagaaagg tcatgaacaa taacgatctc
      361 tttcaggcat cacgtcggcg ttttctggca caactcggca gcttaaccga cgccgggacg
      421 ctggggccgt cattgttaac gccgcgacgt gcgactgcgg cgcaagcggc gactgacgct
      481 tctagaggtt ctcatcatca tcatcatcat ggtatgacta gcatgactgg tggacagcaa
      541 atgggtcggg atctgtacga cgatgatgat aaggacctcg ccacagaatt catggtcgac
      601 tcatcaatga agaggcgctg gaagaaaaac ttcattgccg tcagcgctgc caaccggttc
      661 aagaagatct ccagctccgg ggcactgggg tctgcaggct acaacagcac caacgtctat
      721 atcatggccg acaagcagaa gaacggcatc aaggccaact tcaagatccg ccacaacatc
      781 gaggacggcg gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
      841 cccgtgctgc tgcccgacaa ccactacctg agcttccagt ccgccctgag caaagacccc
      901 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
      961 ggtatggacg agctgtacaa ggtcgacggt ggcagcggtg gcaccggtgt gagcaagggc
     1021 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     1081 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     1141 aagctcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctc
     1201 ggctacggcc tgaagtgctt cgcccgctac cccgaccaca tgaagcagca cgacttcttc
     1261 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     1321 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     1381 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     1441 ggtaccgacc aactgacaga agagcagatt gcagagttca aagaagcctt ctcattattc
     1501 gacaaggatg gggacggcac catcaccaca aaggaacttg gcaccgttat gaggtcgctt
     1561 ggacaaaacc caacggaagc agaattgcag gatatgatca atgaagtcga tgctgatggc
     1621 aatggaacga tatactttcc tgaatttctt actatgatgg ctagaaaaat gaaggacaca
     1681 gacagcgaag aggaaatccg agaagcattc cgtgtttttg acaaggatgg gaacggctac
     1741 atcagcgctg ctcagttacg tcacgtcatg acaaacctcg gggagaagtt aacagatgaa
     1801 gaagttgatg aaatgataag ggaagcagat atcgatggtg atggccaagt aaactatgaa
     1861 gagtttgtac aaatgatgac agcaaagtaa gcgtgcgaat ggtaacaagc ttggctgttt
     1921 tggcggatga gagaagattt tcagcctgat acagattaaa tcagaacgca gaagcggtct
     1981 gataaaacag aatttgcctg gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa
     2041 ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg tctccccatg cgagagtagg
     2101 gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta
     2161 tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga gcggatttga
     2221 acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa actgccaggc
     2281 atcaaattaa gcagaaggcc atcctgacgg atggcctttt tgcgtttcta caaactcttt
     2341 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     2401 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     2461 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     2521 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     2581 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     2641 aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt
     2701 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     2761 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     2821 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     2881 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     2941 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     3001 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     3061 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     3121 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     3181 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     3241 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     3301 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     3361 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     3421 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     3481 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     3541 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     3601 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     3661 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     3721 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     3781 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     3841 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     3901 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     3961 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     4021 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     4081 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     4141 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     4201 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg
     4261 catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc
     4321 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     4381 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     4441 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     4501 cgaaacgcgc gaggcagcag atcaattcgc gcgcgaaggc gaagcggcat gcataatgtg
     4561 cctgtcaaat ggacgaagca gggattctgc aaaccctatg ctactccgtc aagccgtcaa
     4621 ttgtctgatt cgttaccaat tatgacaact tgacggctac atcattcact ttttcttcac
     4681 aaccggcacg gaactcgctc gggctggccc cggtgcattt tttaaatacc cgcgagaaat
     4741 agagttgatc gtcaaaacca acattgcgac cgacggtggc gataggcatc cgggtggtgc
     4801 tcaaaagcag cttcgcctgg ctgatacgtt ggtcctcgcg ccagcttaag acgctaatcc
     4861 ctaactgctg gcggaaaaga tgtgacagac gcgacggcga caagcaaaca tgctgtgcga
     4921 cgctggcgat atcaaaattg ctgtctgcca ggtgatcgct gatgtactga caagcctcgc
     4981 gtacccgatt atccatcggt ggatggagcg actcgttaat cgcttccatg cgccgcagta
     5041 acaattgctc aagcagattt atcgccagca gctccgaata gcgcccttcc ccttgcccgg
     5101 cgttaatgat ttgcccaaac aggtcgctga aatgcggctg gtgcgcttca tccgggcgaa
     5161 agaaccccgt attggcaaat attgacggcc agttaagcca ttcatgccag taggcgcgcg
     5221 gacgaaagta aacccactgg tgataccatt cgcgagcctc cggatgacga ccgtagtgat
     5281 gaatctctcc tggcgggaac agcaaaatat cacccggtcg gcaaacaaat tctcgtccct
     5341 gatttttcac caccccctga ccgcgaatgg tgagattgag aatataacct ttcattccca
     5401 gcggtcggtc gataaaaaaa tcgagataac cgttggcctc aatcggcgtt aaacccgcca
     5461 ccagatgggc attaaacgag tatcccggca gcaggggatc attttgcgct tcagccatac
     5521 ttttcatact cccgccattc agag
//
