LOCUS       sequence-389886-        5687 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Green fluorescent Ca2+ ion indicator for bacterial expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5687)
  AUTHORS   Zhu W, Takeuchi S, Imai S, Terada T, Ueda T, Nasu Y, Terai T, 
            Campbell RE
  TITLE     Chemigenetic indicators based on synthetic chelators and green 
            fluorescent protein.
  JOURNAL   Nat Chem Biol. 2023 Jan;19(1):38-44. doi: 
            10.1038/s41589-022-01134-z. Epub 2022 Sep 22.
  PUBMED    36138142
REFERENCE   2  (bases 1 to 5687)
  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: 
            "10.1038/s41589-022-01134-z"; journalName: "Nat Chem Biol"; date: 
            "2023-01"; volume: "19"; issue: "1"; pages: "38-44"
COMMENT     Sequence Label: pBad-HaloGFP-Ca0.5
FEATURES             Location/Qualifiers
     source          1..5687
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        22..306
                     /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     229..248
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             352..369
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             373..405
                     /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             join(409..417,418..432)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     1086..1107
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1213..1234)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1474..1493)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2109..2126)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2279..2365
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2457..2484
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2503..2594
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2595..2663,2664..3455)
                     /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(2813..2832)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3626..4214
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4115..4134
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4368..4385
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4400..4540
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4466..4485)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4626..4648
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4804..5682)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFEISSVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 tcatactccc gccattcaga gaagaaacca attgtccata ttgcatcaga cattgccgtc
       61 actgcgtctt ttactggctc ttctcgctaa ccaaaccggt aaccccgctt attaaaagca
      121 ttctgtaaca aagcgggacc aaagccatga caaaaacgcg taacaaaagt gtctataatc
      181 acggcagaaa agtccacatt gattatttgc acggcgtcac actttgctat gccatagcat
      241 ttttatccat aagattagcg gatcctacct gacgcttttt atcgcaactc tctactgttt
      301 ctccataccc gttttttggg ctaacaggag gaattaacca tggggggttc tcatcatcat
      361 catcatcatg gtatggctag catgactggt ggacagcaaa tgggtcggga tctgtacgac
      421 gatgacgata aggatccgag ctcgagaatg gcagaaatcg gtactggctt tccattcgac
      481 ccccattatg tggaagtcct gggcgagcgc atgcactacg tcgatgttgg tccgcgcgat
      541 ggcacccctg tgctgttcct gcacggtaac ccgacctcct cctacgtgtg gcgcaacatc
      601 atcccgcatg ttgcaccgac ccatcgctgc attgctccag acctgatcgg tatgggcaaa
      661 tccgacaaac cagacctggg ttatttcttc gacgaccacg tccgcttcat ggatgccttc
      721 atcgaagccc tgggtctgga agaggtcgtc ctggtcattc acgactgggg ctccgctctg
      781 ggtttccact gggccaagcg caatccagag cgcgtcaaag gtattgcatt tatggagttc
      841 atccgcccta tcccgacctg ggacgaatgg ccaggtgaaa acgtctatat caaggccgac
      901 aagcagaaga acggcatcaa ggcgaacttc aagatccgcc acaacatcga ggacggcggc
      961 gtgcaggctg cctaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1021 cccgacaacc acactctgag cgtccagtcc aaactttcga aagaccccaa cgagaagcgc
     1081 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1141 ctgtacaagg gcggtaccgg agggagcatg gtgagcaagg gcgaggagct gttcaccggg
     1201 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
     1261 ggcgagggtg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
     1321 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc
     1381 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     1441 ggctacatcc aggagcgcac catcttcttc ggtgacgacg gcaactacaa gacccgcgcc
     1501 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     1561 aaggaggacg gcaacatcct ggggcataag ctggagtaca accttggcgc tggtggtacc
     1621 ttccaggcct tccgcaccac cgacgtcggc cgcaagctga tcatcgatca gaacgttttt
     1681 atcgagggta cgctgccgat gggtgtcgtc cgcccgctga ctgaagtcga gatggaccat
     1741 taccgcgagc cgttcctgaa tcctgttgac cgcgagccac tgtggcgctt cccaaacgag
     1801 ctgccaatcg ccggtgagcc agcgaacatc gtcgcgctgg tcgaagaata catggactgg
     1861 ctgcaccagt cccctgtccc gaagctgctg ttctggggca ccccaggcgt tctgatccca
     1921 ccggccgaag ccgctcgcct ggccaaaagc ctgcctaact gcaaggctgt ggacatcggc
     1981 ccgggtctga atctgctgca agaagacaac ccggacctga tcggcagcga gatcgcgcgc
     2041 tggctgtcga cgctggagat ttccggctga aagcttggct gttttggcgg atgagagaag
     2101 attttcagcc tgatacagat taaatcagaa cgcagaagcg gtctgataaa acagaatttg
     2161 cctggcggca gtagcgcggt ggtcccacct gaccccatgc cgaactcaga agtgaaacgc
     2221 cgtagcgccg atggtagtgt ggggtctccc catgcgagag tagggaactg ccaggcatca
     2281 aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt
     2341 gaacgctctc ctgagtagga caaatccgcc gggagcggat ttgaacgttg cgaagcaacg
     2401 gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa
     2461 ggccatcctg acggatggcc tttttgcgtt tctacaaact cttttgttta tttttctaaa
     2521 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     2581 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     2641 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     2701 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     2761 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     2821 gcgcggtatt atcccgtgtt gacgccgggc aagagcaact cggtcgccgc atacactatt
     2881 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     2941 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     3001 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     3061 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     3121 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     3181 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     3241 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     3301 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     3361 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     3421 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     3481 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     3541 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     3601 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     3661 tgcaaacaaa acaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     3721 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     3781 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     3841 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     3901 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     3961 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     4021 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     4081 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     4141 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     4201 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     4261 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     4321 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     4381 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     4441 cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     4501 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     4561 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     4621 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     4681 gcagatcaat tcgcgcgcga aggcgaagcg gcatgcataa tgtgcctgtc aaatggacga
     4741 agcagggatt ctgcaaaccc tatgctactc cgtcaagccg tcaattgtct gattcgttac
     4801 caattatgac aacttgacgg ctacatcatt cactttttct tcacaaccgg cacggaactc
     4861 gctcgggctg gccccggtgc attttttaaa tacccgcgag aaatagagtt gatcgtcaaa
     4921 accaacattg cgaccgacgg tggcgatagg catccgggtg gtgctcaaaa gcagcttcgc
     4981 ctggctgata cgttggtcct cgcgccagct taagacgcta atccctaact gctggcggaa
     5041 aagatgtgac agacgcgacg gcgacaagca aacatgctgt gcgacgctgg agatttcaaa
     5101 attgctgtct gccaggtgat cgctgatgta ctgacaagcc tcgcgtaccc gattatccat
     5161 cggtggatgg agcgactcgt taatcgcttc catgcgccgc agtaacaatt gctcaagcag
     5221 atttatcgcc agcagctccg aatagcgccc ttccccttgc ccggcgttaa tgatttgccc
     5281 aaacaggtcg ctgaaatgcg gctggtgcgc ttcatccggg cgaaagaacc ccgtattggc
     5341 aaatattgac ggccagttaa gccattcatg ccagtaggcg cgcggacgaa agtaaaccca
     5401 ctggtgatac cattcgcgag cctccggatg acgaccgtag tgatgaatct ctcctggcgg
     5461 gaacagcaaa atatcacccg gtcggcaaac aaattctcgt ccctgatttt tcaccacccc
     5521 ctgaccgcga atggtgagat tgagaatata acctttcatt cccagcggtc ggtcgataaa
     5581 aaaatcgaga taaccgttgg cctcaatcgg cgttaaaccc gccaccagat gggcattaaa
     5641 cgagtatccc ggcagcaggg gatcattttg cgcttcagcc atacttt
//
