LOCUS       sequence-389861-        5681 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 5681)
  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 5681)
  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-151-154
FEATURES             Location/Qualifiers
     source          1..5681
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        18..302
                     /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     225..244
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             348..365
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             369..401
                     /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(405..413,414..428)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     1106..1127
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1233..1254)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1494..1513)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2099..2116)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2269..2355
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2447..2474
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2493..2584
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2585..2653,2654..3445)
                     /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(2803..2822)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3616..4204
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4105..4124
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4358..4375
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4390..4530
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4456..4475)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4616..4638
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4794..5672)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 actcccgcca ttcagagaag aaaccaattg tccatattgc atcagacatt gccgtcactg
       61 cgtcttttac tggctcttct cgctaaccaa accggtaacc ccgcttatta aaagcattct
      121 gtaacaaagc gggaccaaag ccatgacaaa aacgcgtaac aaaagtgtct ataatcacgg
      181 cagaaaagtc cacattgatt atttgcacgg cgtcacactt tgctatgcca tagcattttt
      241 atccataaga ttagcggatc ctacctgacg ctttttatcg caactctcta ctgtttctcc
      301 atacccgttt tttgggctaa caggaggaat taaccatggg gggttctcat catcatcatc
      361 atcatggtat ggctagcatg actggtggac agcaaatggg tcgggatctg tacgacgatg
      421 acgataagga tccgagctcg agaatggcag aaatcggtac tggctttcca ttcgaccccc
      481 attatgtgga agtcctgggc gagcgcatgc actacgtcga tgttggtccg cgcgatggca
      541 cccctgtgct gttcctgcac ggtaacccga cctcctccta cgtgtggcgc aacatcatcc
      601 cgcatgttgc accgacccat cgctgcattg ctccagacct gatcggtatg ggcaaatccg
      661 acaaaccaga cctgggttat ttcttcgacg accacgtccg cttcatggat gccttcatcg
      721 aagccctggg tctggaagag gtcgtcctgg tcattcacga ctggggctcc gctctgggtt
      781 tccactgggc caagcgcaat ccagagcgcg tcaaaggtat tgcatttatg gagttcatcc
      841 gccctatccc gacctgggac gaatggccag aatttgcccg cgagaccttc cagggtgaaa
      901 acgtctatat caaggccgac aagcagaaga acggcatcaa ggcgaacttc aagatccgcc
      961 acaacatcga ggacggcggc gtgcagctcg cctaccacta ccagcagaac acccccatcg
     1021 gcgacggccc cgtgctgctg cccgacaacc actacctgag cgtccagtcc aaactttcga
     1081 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     1141 tcactctcgg catggacgag ctgtacaagg gcggtaccgg agggagcatg gtgagcaagg
     1201 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     1261 gccacaagtt cagcgtgtcc ggcgagggtg agggcgatgc cacctacggc aagctgaccc
     1321 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     1381 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
     1441 tcaagtccgc catgcccgaa ggctacatcc aggagcgcac catcttcttc aaggacgacg
     1501 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     1561 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcataag ctggagtaca
     1621 accttggcac cgacgtcggc cgcaagctga tcatcgatca gaacgttttt atcgagggta
     1681 cgctgccgat gggtgtcgtc cgcccgctga ctgaagtcga gatggaccat taccgcgagc
     1741 cgttcctgaa tcctgttgac cgcgagccac tgtggcgctt cccaaacgag ctgccaatcg
     1801 ccggtgagcc agcgaacatc gtcgcgctgg tcgaagaata catggactgg ctgcaccagt
     1861 cccctgtccc gaagctgctg ttctggggca ccccaggcgt tctgatccca ccggccgaag
     1921 ccgctcgcct ggccaaaagc ctgcctaact gcaaggctgt ggacatcggc ccgggtctga
     1981 atctgctgca agaagacaac ccggacctga tcggcagcga gatcgcgcgc tggctgtcga
     2041 cgctcgagat ttccggctga aagcttggct gttttggcgg atgagagaag attttcagcc
     2101 tgatacagat taaatcagaa cgcagaagcg gtctgataaa acagaatttg cctggcggca
     2161 gtagcgcggt ggtcccacct gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg
     2221 atggtagtgt ggggtctccc catgcgagag tagggaactg ccaggcatca aataaaacga
     2281 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc
     2341 ctgagtagga caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg
     2401 tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg
     2461 acggatggcc tttttgcgtt tctacaaact cttttgttta tttttctaaa tacattcaaa
     2521 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     2581 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     2641 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     2701 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     2761 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     2821 atcccgtgtt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     2881 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     2941 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     3001 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     3061 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     3121 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     3181 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     3241 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     3301 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     3361 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     3421 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     3481 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     3541 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     3601 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     3661 acaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     3721 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     3781 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     3841 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     3901 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     3961 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     4021 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     4081 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     4141 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     4201 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     4261 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     4321 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     4381 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     4441 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     4501 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     4561 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     4621 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gcagatcaat
     4681 tcgcgcgcga aggcgaagcg gcatgcataa tgtgcctgtc aaatggacga agcagggatt
     4741 ctgcaaaccc tatgctactc cgtcaagccg tcaattgtct gattcgttac caattatgac
     4801 aacttgacgg ctacatcatt cactttttct tcacaaccgg cacggaactc gctcgggctg
     4861 gccccggtgc attttttaaa tacccgcgag aaatagagtt gatcgtcaaa accaacattg
     4921 cgaccgacgg tggcgatagg catccgggtg gtgctcaaaa gcagcttcgc ctggctgata
     4981 cgttggtcct cgcgccagct taagacgcta atccctaact gctggcggaa aagatgtgac
     5041 agacgcgacg gcgacaagca aacatgctgt gcgacgctgg cgatatcaaa attgctgtct
     5101 gccaggtgat cgctgatgta ctgacaagcc tcgcgtaccc gattatccat cggtggatgg
     5161 agcgactcgt taatcgcttc catgcgccgc agtaacaatt gctcaagcag atttatcgcc
     5221 agcagctccg aatagcgccc ttccccttgc ccggcgttaa tgatttgccc aaacaggtcg
     5281 ctgaaatgcg gctggtgcgc ttcatccggg cgaaagaacc ccgtattggc aaatattgac
     5341 ggccagttaa gccattcatg ccagtaggcg cgcggacgaa agtaaaccca ctggtgatac
     5401 cattcgcgag cctccggatg acgaccgtag tgatgaatct ctcctggcgg gaacagcaaa
     5461 atatcacccg gtcggcaaac aaattctcgt ccctgatttt tcaccacccc ctgaccgcga
     5521 atggtgagat tgagaatata acctttcatt cccagcggtc ggtcgataaa aaaatcgaga
     5581 taaccgttgg cctcaatcgg cgttaaaccc gccaccagat gggcattaaa cgagtatccc
     5641 ggcagcaggg gatcattttg cgcttcagcc atacttttca t
//
