LOCUS       sequence-389866-        5684 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 5684)
  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 5684)
  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-153-155
FEATURES             Location/Qualifiers
     source          1..5684
                     /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     1116..1137
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1243..1264)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1504..1523)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2106..2123)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2276..2362
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2454..2481
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2500..2591
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2592..2660,2661..3452)
                     /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(2810..2829)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3623..4211
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4112..4131
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4365..4382
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4397..4537
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4463..4482)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4623..4645
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4801..5679)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     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 ccagaatttg cccgcgagac cttccaggcc
      901 ttcggtgaaa acgtctatat caaggccgac aagcagaaga acggcatcaa ggcgaacttc
      961 aagatccgcc acaacatcga ggacggcggc gtgcagctcg cctaccacta ccagcagaac
     1021 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cgtccagtcc
     1081 aaactttcga aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     1141 gccgccggga tcactctcgg catggacgag ctgtacaagg gcggtaccgg agggagcatg
     1201 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     1261 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggtg agggcgatgc cacctacggc
     1321 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     1381 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     1441 cacgacttct tcaagtccgc catgcccgaa ggctacatcc aggagcgcac catcttcttc
     1501 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     1561 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcataag
     1621 ctggagtaca accttggcga cgtcggccgc aagctgatca tcgatcagaa cgtttttatc
     1681 gagggtacgc tgccgatggg tgtcgtccgc ccgctgactg aagtcgagat ggaccattac
     1741 cgcgagccgt tcctgaatcc tgttgaccgc gagccactgt ggcgcttccc aaacgagctg
     1801 ccaatcgccg gtgagccagc gaacatcgtc gcgctggtcg aagaatacat ggactggctg
     1861 caccagtccc ctgtcccgaa gctgctgttc tggggcaccc caggcgttct gatcccaccg
     1921 gccgaagccg ctcgcctggc caaaagcctg cctaactgca aggctgtgga catcggcccg
     1981 ggtctgaatc tgctgcaaga agacaacccg gacctgatcg gcagcgagat cgcgcgctgg
     2041 ctgtcgacgc tcgagatttc cggctgaaag cttggctgtt ttggcggatg agagaagatt
     2101 ttcagcctga tacagattaa atcagaacgc agaagcggtc tgataaaaca gaatttgcct
     2161 ggcggcagta gcgcggtggt cccacctgac cccatgccga actcagaagt gaaacgccgt
     2221 agcgccgatg gtagtgtggg gtctccccat gcgagagtag ggaactgcca ggcatcaaat
     2281 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa
     2341 cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc
     2401 cggagggtgg cgggcaggac gcccgccata aactgccagg catcaaatta agcagaaggc
     2461 catcctgacg gatggccttt ttgcgtttct acaaactctt ttgtttattt ttctaaatac
     2521 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     2581 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     2641 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     2701 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     2761 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     2821 cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     2881 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     2941 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     3001 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     3061 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     3121 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     3181 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     3241 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     3301 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     3361 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     3421 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     3481 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     3541 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     3601 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     3661 aaacaaaaca accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     3721 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     3781 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     3841 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     3901 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     3961 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     4021 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     4081 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     4141 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     4201 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     4261 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     4321 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     4381 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     4441 accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     4501 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     4561 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     4621 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagca
     4681 gatcaattcg cgcgcgaagg cgaagcggca tgcataatgt gcctgtcaaa tggacgaagc
     4741 agggattctg caaaccctat gctactccgt caagccgtca attgtctgat tcgttaccaa
     4801 ttatgacaac ttgacggcta catcattcac tttttcttca caaccggcac ggaactcgct
     4861 cgggctggcc ccggtgcatt ttttaaatac ccgcgagaaa tagagttgat cgtcaaaacc
     4921 aacattgcga ccgacggtgg cgataggcat ccgggtggtg ctcaaaagca gcttcgcctg
     4981 gctgatacgt tggtcctcgc gccagcttaa gacgctaatc cctaactgct ggcggaaaag
     5041 atgtgacaga cgcgacggcg acaagcaaac atgctgtgcg acgctggcga tatcaaaatt
     5101 gctgtctgcc aggtgatcgc tgatgtactg acaagcctcg cgtacccgat tatccatcgg
     5161 tggatggagc gactcgttaa tcgcttccat gcgccgcagt aacaattgct caagcagatt
     5221 tatcgccagc agctccgaat agcgcccttc cccttgcccg gcgttaatga tttgcccaaa
     5281 caggtcgctg aaatgcggct ggtgcgcttc atccgggcga aagaaccccg tattggcaaa
     5341 tattgacggc cagttaagcc attcatgcca gtaggcgcgc ggacgaaagt aaacccactg
     5401 gtgataccat tcgcgagcct ccggatgacg accgtagtga tgaatctctc ctggcgggaa
     5461 cagcaaaata tcacccggtc ggcaaacaaa ttctcgtccc tgatttttca ccaccccctg
     5521 accgcgaatg gtgagattga gaatataacc tttcattccc agcggtcggt cgataaaaaa
     5581 atcgagataa ccgttggcct caatcggcgt taaacccgcc accagatggg cattaaacga
     5641 gtatcccggc agcaggggat cattttgcgc ttcagccata cttt
//
