LOCUS       sequence-389846-        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-159-160
FEATURES             Location/Qualifiers
     source          1..5687
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        27..311
                     /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     234..253
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             357..374
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             378..410
                     /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(414..422,423..437)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     1139..1160
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1266..1287)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1527..1546)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2114..2131)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2284..2370
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2462..2489
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2508..2599
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2600..2668,2669..3460)
                     /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(2818..2837)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3631..4219
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4120..4139
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4373..4390
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4405..4545
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4471..4490)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4631..4653
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4809..5687)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 acttttcata ctcccgccat tcagagaaga aaccaattgt ccatattgca tcagacattg
       61 ccgtcactgc gtcttttact ggctcttctc gctaaccaaa ccggtaaccc cgcttattaa
      121 aagcattctg taacaaagcg ggaccaaagc catgacaaaa acgcgtaaca aaagtgtcta
      181 taatcacggc agaaaagtcc acattgatta tttgcacggc gtcacacttt gctatgccat
      241 agcattttta tccataagat tagcggatcc tacctgacgc tttttatcgc aactctctac
      301 tgtttctcca tacccgtttt ttgggctaac aggaggaatt aaccatgggg ggttctcatc
      361 atcatcatca tcatggtatg gctagcatga ctggtggaca gcaaatgggt cgggatctgt
      421 acgacgatga cgataaggat ccgagctcga gaatggcaga aatcggtact ggctttccat
      481 tcgaccccca ttatgtggaa gtcctgggcg agcgcatgca ctacgtcgat gttggtccgc
      541 gcgatggcac ccctgtgctg ttcctgcacg gtaacccgac ctcctcctac gtgtggcgca
      601 acatcatccc gcatgttgca ccgacccatc gctgcattgc tccagacctg atcggtatgg
      661 gcaaatccga caaaccagac ctgggttatt tcttcgacga ccacgtccgc ttcatggatg
      721 ccttcatcga agccctgggt ctggaagagg tcgtcctggt cattcacgac tggggctccg
      781 ctctgggttt ccactgggcc aagcgcaatc cagagcgcgt caaaggtatt gcatttatgg
      841 agttcatccg ccctatcccg acctgggacg aatggccaga atttgcccgc gagaccttcc
      901 aggccttccg caccaccgac gtcggcggtg aaaacgtcta tatcaaggcc gacaagcaga
      961 agaacggcat caaggcgaac ttcaagatcc gccacaacat cgaggacggc ggcgtgcagc
     1021 tcgcctacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     1081 accactacct gagcgtccag tccaaacttt cgaaagaccc caacgagaag cgcgatcaca
     1141 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     1201 agggcggtac cggagggagc atggtgagca agggcgagga gctgttcacc ggggtggtgc
     1261 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
     1321 gtgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     1381 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
     1441 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctaca
     1501 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     1561 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     1621 acggcaacat cctggggcat aagctggagt acaaccttgg cctgatcatc gatcagaacg
     1681 tttttatcga gggtacgctg ccgatgggtg tcgtccgccc gctgactgaa gtcgagatgg
     1741 accattaccg cgagccgttc ctgaatcctg ttgaccgcga gccactgtgg cgcttcccaa
     1801 acgagctgcc aatcgccggt gagccagcga acatcgtcgc gctggtcgaa gaatacatgg
     1861 actggctgca ccagtcccct gtcccgaagc tgctgttctg gggcacccca ggcgttctga
     1921 tcccaccggc cgaagccgct cgcctggcca aaagcctgcc taactgcaag gctgtggaca
     1981 tcggcccggg tctgaatctg ctgcaagaag acaacccgga cctgatcggc agcgagatcg
     2041 cgcgctggct gtcgacgctc gagatttccg gctgaaagct tggctgtttt ggcggatgag
     2101 agaagatttt cagcctgata cagattaaat cagaacgcag aagcggtctg ataaaacaga
     2161 atttgcctgg cggcagtagc gcggtggtcc cacctgaccc catgccgaac tcagaagtga
     2221 aacgccgtag cgccgatggt agtgtggggt ctccccatgc gagagtaggg aactgccagg
     2281 catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg
     2341 tcggtgaacg ctctcctgag taggacaaat ccgccgggag cggatttgaa cgttgcgaag
     2401 caacggcccg gagggtggcg ggcaggacgc ccgccataaa ctgccaggca tcaaattaag
     2461 cagaaggcca tcctgacgga tggccttttt gcgtttctac aaactctttt gtttattttt
     2521 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     2581 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     2641 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     2701 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     2761 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     2821 atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca
     2881 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     2941 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     3001 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     3061 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     3121 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     3181 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     3241 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     3301 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     3361 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     3421 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     3481 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     3541 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     3601 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     3661 ctgcttgcaa acaaaacaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     3721 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     3781 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     3841 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     3901 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     3961 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     4021 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     4081 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     4141 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     4201 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     4261 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     4321 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     4381 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     4441 tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     4501 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     4561 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     4621 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     4681 aggcagcaga tcaattcgcg cgcgaaggcg aagcggcatg cataatgtgc ctgtcaaatg
     4741 gacgaagcag ggattctgca aaccctatgc tactccgtca agccgtcaat tgtctgattc
     4801 gttaccaatt atgacaactt gacggctaca tcattcactt tttcttcaca accggcacgg
     4861 aactcgctcg ggctggcccc ggtgcatttt ttaaataccc gcgagaaata gagttgatcg
     4921 tcaaaaccaa cattgcgacc gacggtggcg ataggcatcc gggtggtgct caaaagcagc
     4981 ttcgcctggc tgatacgttg gtcctcgcgc cagcttaaga cgctaatccc taactgctgg
     5041 cggaaaagat gtgacagacg cgacggcgac aagcaaacat gctgtgcgac gctggcgata
     5101 tcaaaattgc tgtctgccag gtgatcgctg atgtactgac aagcctcgcg tacccgatta
     5161 tccatcggtg gatggagcga ctcgttaatc gcttccatgc gccgcagtaa caattgctca
     5221 agcagattta tcgccagcag ctccgaatag cgcccttccc cttgcccggc gttaatgatt
     5281 tgcccaaaca ggtcgctgaa atgcggctgg tgcgcttcat ccgggcgaaa gaaccccgta
     5341 ttggcaaata ttgacggcca gttaagccat tcatgccagt aggcgcgcgg acgaaagtaa
     5401 acccactggt gataccattc gcgagcctcc ggatgacgac cgtagtgatg aatctctcct
     5461 ggcgggaaca gcaaaatatc acccggtcgg caaacaaatt ctcgtccctg atttttcacc
     5521 accccctgac cgcgaatggt gagattgaga atataacctt tcattcccag cggtcggtcg
     5581 ataaaaaaat cgagataacc gttggcctca atcggcgtta aacccgccac cagatgggca
     5641 ttaaacgagt atcccggcag caggggatca ttttgcgctt cagccat
//
