LOCUS       sequence-89444-e        5489 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encoded a yellow fluorescent calcium ion indicator with inverted 
            fluorescence response for expression in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5489)
  AUTHORS   Zhao Y, Abdelfattah AS, Zhao Y, Ruangkittisakul A, Ballanyi K, 
            Campbell RE, Harrison DJ
  TITLE     Microfluidic cell sorter-aided directed evolution of a protein-based
            calcium ion indicator with an inverted fluorescent response.
  JOURNAL   Integr Biol (Camb). 2014 May 19.
  PUBMED    24840546
REFERENCE   2  (bases 1 to 5489)
  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: "Integr Biol
            (Camb). 2014 May 19."
COMMENT     Sequence Label: pTorPE-Y-GECO1
FEATURES             Location/Qualifiers
     source          1..5489
                     /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="MASMTGGQQMG"
     CDS             601..657
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     CDS             1611..1625
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     primer_bind     complement(1890..1907)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2060..2146
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2238..2265
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2284..2375
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2376..2444,2445..3236)
                     /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(2594..2613)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3407..3995
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3896..3915
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4149..4166
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4181..4321
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4247..4266)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4407..4429
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4585..5463)
                     /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 ggtatggcta gcatgactgg tggacagcaa
      541 atgggtcggg atctgtacga cgaagatgat aaggatctcg ccacaatggc cgactcatca
      601 cgtcgtaagt ggaataagac aggtcacgca gtcagagcta taggccggct gagctcactc
      661 gagggttata gtaccgagaa aattgtgcct gttccaaagc aagggatatt aaaaggaaag
      721 gtcagcatgt acctattgct gaaggatggc ggtcgttatc attgccagtt tgatacggta
      781 tataaagcta aatcagtgcc ctccaagatg ccaggacggc attttataca gcataagctc
      841 cttagggagg accgctctga tgctaaaaac cataaatggc aattaacaga acatgcaatc
      901 gcaggcatgg acgagctgta caagggaggt acaggcggga gtatggtgag caagggcgag
      961 gggcaatcga agcatgggct aaaggaggag atgacggtga agtaccacat ggaaggttgc
     1021 gttaatggtc acaaatttgt cattactgga gagggcattg gaaacccttt taagggtaag
     1081 caaactgcaa atttgtgtgt gatagaagga ggcccgctgc cgttctcgga ggacatttta
     1141 agtccgggtt ttaaatatgg tgaccggatt ttcacagagt acccacagga tattgtagat
     1201 tacttcaaga actcatgtcc agagggctat acgtgggaaa ggagcttctt ctatgaggac
     1261 ggagcggtct gtcgatgcaa cgcggacata acagtctctg aaaaggagaa ctgcatctat
     1321 cacaaaagta tcttctatgg ggtgaatttt cccgccgacg gccccgtaat gaagaaaatg
     1381 accactaatt gggaagctga cacgcgtgac caactgactg aagagcagat cgcagaattt
     1441 aaagaggctt tctccctatt tgacaaggac ggggatggga cgatgacaac cgaggagctg
     1501 gggacggtgc tgcggtctct ggggcagaac ccctcagaag cagagctgcg ggacatgatc
     1561 aatgaagtag atgccgacgg tgacggcaca atcggcttcc ctgagttcct gacgatgatg
     1621 acaagaaaaa tgaatgacac ggacagtgaa gaggaaatta gagaagcgtt ccgtgtgttt
     1681 gataaggacg gcaatggcta catcggcgca gcagagcttc gccacgtgat gacaaacctt
     1741 ggagagaagt taacagatga agagtttgat gaaatgatca gggtagcaga ctccgacggg
     1801 gatggtcagg taaactacga agagtttgta caaatgatga cagcaaagtg aaagcttggc
     1861 tgttttggcg gatgagagaa gattttcagc ctgatacaga ttaaatcaga acgcagaagc
     1921 ggtctgataa aacagaattt gcctggcggc agtagcgcgg tggtcccacc tgaccccatg
     1981 ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg tggggtctcc ccatgcgaga
     2041 gtagggaact gccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg
     2101 ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgggagcgga
     2161 tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc cataaactgc
     2221 caggcatcaa attaagcaga aggccatcct gacggatggc ctttttgcgt ttctacaaac
     2281 tcttttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     2341 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     2401 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     2461 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     2521 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     2581 cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac
     2641 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     2701 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     2761 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     2821 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     2881 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     2941 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     3001 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     3061 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     3121 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     3181 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     3241 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     3301 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     3361 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     3421 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     3481 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     3541 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     3601 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     3661 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     3721 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     3781 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     3841 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     3901 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     3961 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     4021 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     4081 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     4141 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
     4201 ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg
     4261 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     4321 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     4381 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     4441 atcaccgaaa cgcgcgaggc agcagatcaa ttcgcgcgcg aaggcgaagc ggcatgcata
     4501 atgtgcctgt caaatggacg aagcagggat tctgcaaacc ctatgctact ccgtcaagcc
     4561 gtcaattgtc tgattcgtta ccaattatga caacttgacg gctacatcat tcactttttc
     4621 ttcacaaccg gcacggaact cgctcgggct ggccccggtg cattttttaa atacccgcga
     4681 gaaatagagt tgatcgtcaa aaccaacatt gcgaccgacg gtggcgatag gcatccgggt
     4741 ggtgctcaaa agcagcttcg cctggctgat acgttggtcc tcgcgccagc ttaagacgct
     4801 aatccctaac tgctggcgga aaagatgtga cagacgcgac ggcgacaagc aaacatgctg
     4861 tgcgacgctg gcgatatcaa aattgctgtc tgccaggtga tcgctgatgt actgacaagc
     4921 ctcgcgtacc cgattatcca tcggtggatg gagcgactcg ttaatcgctt ccatgcgccg
     4981 cagtaacaat tgctcaagca gatttatcgc cagcagctcc gaatagcgcc cttccccttg
     5041 cccggcgtta atgatttgcc caaacaggtc gctgaaatgc ggctggtgcg cttcatccgg
     5101 gcgaaagaac cccgtattgg caaatattga cggccagtta agccattcat gccagtaggc
     5161 gcgcggacga aagtaaaccc actggtgata ccattcgcga gcctccggat gacgaccgta
     5221 gtgatgaatc tctcctggcg ggaacagcaa aatatcaccc ggtcggcaaa caaattctcg
     5281 tccctgattt ttcaccaccc cctgaccgcg aatggtgaga ttgagaatat aacctttcat
     5341 tcccagcggt cggtcgataa aaaaatcgag ataaccgttg gcctcaatcg gcgttaaacc
     5401 cgccaccaga tgggcattaa acgagtatcc cggcagcagg ggatcatttt gcgcttcagc
     5461 catacttttc atactcccgc cattcagag
//
