LOCUS       sequence-211258-        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        711..995
                     /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     918..937
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     RBS             1037..1048
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1203..1220
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1224..1256
                     /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(1260..1268,1269..1283)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             1320..1397
                     /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     1628..1649
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1761..1782)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2022..2041)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2655..2672)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2825..2911
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3003..3030
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3049..3140
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3141..3209,3210..4001)
                     /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(3359..3378)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4172..4760
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4661..4680
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4914..4931
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4946..5086
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5012..5031)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5172..5194
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(join(5350..5544,1..684))
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 ctcgcgccag cttaagacgc taatccctaa ctgctggcgg aaaagatgtg acagacgcga
       61 cggcgacaag caaacatgct gtgcgacgct ggcgatatca aaattgctgt ctgccaggtg
      121 atcgctgatg tactgacaag cctcgcgtac ccgattatcc atcggtggat ggagcgactc
      181 gttaatcgct tccatgcgcc gcagtaacaa ttgctcaagc agatttatcg ccagcagctc
      241 cgaatagcgc ccttcccctt gcccggcgtt aatgatttgc ccaaacaggt cgctgaaatg
      301 cggctggtgc gcttcatccg ggcgaaagaa ccccgtattg gcaaatattg acggccagtt
      361 aagccattca tgccagtagg cgcgcggacg aaagtaaacc cactggtgat accattcgcg
      421 agcctccgga tgacgaccgt agtgatgaat ctctcctggc gggaacagca aaatatcacc
      481 cggtcggcaa acaaattctc gtccctgatt tttcaccacc ccctgaccgc gaatggtgag
      541 attgagaata taacctttca ttcccagcgg tcggtcgata aaaaaatcga gataaccgtt
      601 ggcctcaatc ggcgttaaac ccgccaccag atgggcatta aacgagtatc ccggcagcag
      661 gggatcattt tgcgcttcag ccatactttt catactcccg ccattcagag aagaaaccaa
      721 ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct tctcgctaac
      781 caaaccggta accccgctta ttaaaagcat tctgtaacaa agcgggacca aagccatgac
      841 aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg attatttgca
      901 cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg atcctacctg
      961 acgcttttta tcgcaactct ctactgtttc tccatacccg ttttttgggc taacaggagg
     1021 aattaaccat gggtttaaag aggagaaagg tcatgaacaa taacgatctc tttcaggcat
     1081 cacgtcggcg ttttctggca caactcggca gcttaaccga cgccgggacg ctggggccgt
     1141 cattgttaac gccgcgacgt gcgactgcgg cgcaagcggc gactgacgct tctagaggtt
     1201 ctcatcatca tcatcatcat ggtatgacta gcatgactgg tggacagcaa atgggtcggg
     1261 atctgtacga cgatgatgat aaggacctcg ccacagaatt catggtcgac tcatcaatga
     1321 agaggcgctg gaagaaaaac ttcattgccg tcagcgctgc caaccggttc aagaagatct
     1381 ccagctccgg ggcactgggg tctgcaggct acaacagcac caacgtctat atcatggccg
     1441 acaagcagaa gaacggcatc aaggccaact tcaagatccg ccacaacatc gaggacggcg
     1501 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     1561 tgcccgacaa ccactacctg agcttccagt ccgccctgag caaagacccc aacgagaagc
     1621 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggtatggacg
     1681 agctgtacaa ggtcgacggt ggcagcggtg gcaccggtgt gagcaagggc gaggagctgt
     1741 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     1801 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagctcatct
     1861 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctc ggctacggcc
     1921 tgaagtgctt cgcccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     1981 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
     2041 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     2101 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac ggtaccgacc
     2161 aactgacaga agagcagatt gcagagttca aagaagcctt ctcattattc gacaaggatg
     2221 gggacggcac catcaccaca aaggaacttg gcaccgttat gaggtcgctt ggacaaaacc
     2281 caacggaagc agaattgcag gatatgatca atgaagtcga tgctgatggc aatggaacga
     2341 tatactttcc tgaatttctt actatgatgg ctagaaaaat gaaggacaca gacagcgaag
     2401 aggaaatccg agaagcattc cgtgtttttg acaaggatgg gaacggctac atcagcgctg
     2461 ctcagttacg tcacgtcatg acaaacctcg gggagaagtt aacagatgaa gaagttgatg
     2521 aaatgataag ggaagcagat atcgatggtg atggccaagt aaactatgaa gagtttgtac
     2581 aaatgatgac agcaaagtaa gcgtgcgaat ggtaacaagc ttggctgttt tggcggatga
     2641 gagaagattt tcagcctgat acagattaaa tcagaacgca gaagcggtct gataaaacag
     2701 aatttgcctg gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg
     2761 aaacgccgta gcgccgatgg tagtgtgggg tctccccatg cgagagtagg gaactgccag
     2821 gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt
     2881 gtcggtgaac gctctcctga gtaggacaaa tccgccggga gcggatttga acgttgcgaa
     2941 gcaacggccc ggagggtggc gggcaggacg cccgccataa actgccaggc atcaaattaa
     3001 gcagaaggcc atcctgacgg atggcctttt tgcgtttcta caaactcttt tgtttatttt
     3061 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     3121 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     3181 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     3241 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     3301 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     3361 tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac
     3421 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     3481 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     3541 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     3601 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     3661 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     3721 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     3781 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     3841 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     3901 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     3961 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     4021 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     4081 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     4141 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     4201 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     4261 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     4321 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     4381 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     4441 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     4501 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     4561 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     4621 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     4681 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     4741 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     4801 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     4861 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     4921 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     4981 gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     5041 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     5101 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     5161 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     5221 gaggcagcag atcaattcgc gcgcgaaggc gaagcggcat gcataatgtg cctgtcaaat
     5281 ggacgaagca gggattctgc aaaccctatg ctactccgtc aagccgtcaa ttgtctgatt
     5341 cgttaccaat tatgacaact tgacggctac atcattcact ttttcttcac aaccggcacg
     5401 gaactcgctc gggctggccc cggtgcattt tttaaatacc cgcgagaaat agagttgatc
     5461 gtcaaaacca acattgcgac cgacggtggc gataggcatc cgggtggtgc tcaaaagcag
     5521 cttcgcctgg ctgatacgtt ggtc
//
