LOCUS       sequence-389872-        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-177-178
FEATURES             Location/Qualifiers
     source          1..5687
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        17..301
                     /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     224..243
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             347..364
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             368..400
                     /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(404..412,413..427)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     1183..1204
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1310..1331)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1571..1590)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2104..2121)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2274..2360
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2452..2479
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2498..2589
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2590..2658,2659..3450)
                     /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(2808..2827)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3621..4209
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4110..4129
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4363..4380
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4395..4535
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4461..4480)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4621..4643
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4799..5677)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 ctcccgccat tcagagaaga aaccaattgt ccatattgca tcagacattg ccgtcactgc
       61 gtcttttact ggctcttctc gctaaccaaa ccggtaaccc cgcttattaa aagcattctg
      121 taacaaagcg ggaccaaagc catgacaaaa acgcgtaaca aaagtgtcta taatcacggc
      181 agaaaagtcc acattgatta tttgcacggc gtcacacttt gctatgccat agcattttta
      241 tccataagat tagcggatcc tacctgacgc tttttatcgc aactctctac tgtttctcca
      301 tacccgtttt ttgggctaac aggaggaatt aaccatgggg ggttctcatc atcatcatca
      361 tcatggtatg gctagcatga ctggtggaca gcaaatgggt cgggatctgt acgacgatga
      421 cgataaggat ccgagctcga gaatggcaga aatcggtact ggctttccat tcgaccccca
      481 ttatgtggaa gtcctgggcg agcgcatgca ctacgtcgat gttggtccgc gcgatggcac
      541 ccctgtgctg ttcctgcacg gtaacccgac ctcctcctac gtgtggcgca acatcatccc
      601 gcatgttgca ccgacccatc gctgcattgc tccagacctg atcggtatgg gcaaatccga
      661 caaaccagac ctgggttatt tcttcgacga ccacgtccgc ttcatggatg ccttcatcga
      721 agccctgggt ctggaagagg tcgtcctggt cattcacgac tggggctccg ctctgggttt
      781 ccactgggcc aagcgcaatc cagagcgcgt caaaggtatt gcatttatgg agttcatccg
      841 ccctatcccg acctgggacg aatggccaga atttgcccgc gagaccttcc aggccttccg
      901 caccaccgac gtcggccgca agctgatcat cgatcagaac gtttttatcg agggtacgct
      961 gccgatgggt ggtgaaaacg tctatatcaa ggccgacaag cagaagaacg gcatcaaggc
     1021 gaacttcaag atccgccaca acatcgagga cggcggcgtg cagctcgcct accactacca
     1081 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcgt
     1141 ccagtccaaa ctttcgaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     1201 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagggcg gtaccggagg
     1261 gagcatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
     1321 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggtgagg gcgatgccac
     1381 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     1441 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
     1501 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacatccagg agcgcaccat
     1561 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     1621 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     1681 gcataagctg gagtacaacc ttggccgccc gctgactgaa gtcgagatgg accattaccg
     1741 cgagccgttc ctgaatcctg ttgaccgcga gccactgtgg cgcttcccaa acgagctgcc
     1801 aatcgccggt gagccagcga acatcgtcgc gctggtcgaa gaatacatgg actggctgca
     1861 ccagtcccct gtcccgaagc tgctgttctg gggcacccca ggcgttctga tcccaccggc
     1921 cgaagccgct cgcctggcca aaagcctgcc taactgcaag gctgtggaca tcggcccggg
     1981 tctgaatctg ctgcaagaag acaacccgga cctgatcggc agcgagatcg cgcgctggct
     2041 gtcgacgctc gagatttccg gctgaaagct tggctgtttt ggcggatgag agaagatttt
     2101 cagcctgata cagattaaat cagaacgcag aagcggtctg ataaaacaga atttgcctgg
     2161 cggcagtagc gcggtggtcc cacctgaccc catgccgaac tcagaagtga aacgccgtag
     2221 cgccgatggt agtgtggggt ctccccatgc gagagtaggg aactgccagg catcaaataa
     2281 aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg
     2341 ctctcctgag taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg
     2401 gagggtggcg ggcaggacgc ccgccataaa ctgccaggca tcaaattaag cagaaggcca
     2461 tcctgacgga tggccttttt gcgtttctac aaactctttt gtttattttt ctaaatacat
     2521 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     2581 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     2641 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     2701 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     2761 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     2821 gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     2881 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     2941 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     3001 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     3061 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     3121 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     3181 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     3241 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     3301 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     3361 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     3421 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     3481 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     3541 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     3601 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     3661 acaaaacaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     3721 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     3781 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     3841 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     3901 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     3961 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     4021 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     4081 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     4141 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     4201 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     4261 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     4321 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     4381 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     4441 cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
     4501 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
     4561 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     4621 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagcaga
     4681 tcaattcgcg cgcgaaggcg aagcggcatg cataatgtgc ctgtcaaatg gacgaagcag
     4741 ggattctgca aaccctatgc tactccgtca agccgtcaat tgtctgattc gttaccaatt
     4801 atgacaactt gacggctaca tcattcactt tttcttcaca accggcacgg aactcgctcg
     4861 ggctggcccc ggtgcatttt ttaaataccc gcgagaaata gagttgatcg tcaaaaccaa
     4921 cattgcgacc gacggtggcg ataggcatcc gggtggtgct caaaagcagc ttcgcctggc
     4981 tgatacgttg gtcctcgcgc cagcttaaga cgctaatccc taactgctgg cggaaaagat
     5041 gtgacagacg cgacggcgac aagcaaacat gctgtgcgac gctggcgata tcaaaattgc
     5101 tgtctgccag gtgatcgctg atgtactgac aagcctcgcg tacccgatta tccatcggtg
     5161 gatggagcga ctcgttaatc gcttccatgc gccgcagtaa caattgctca agcagattta
     5221 tcgccagcag ctccgaatag cgcccttccc cttgcccggc gttaatgatt tgcccaaaca
     5281 ggtcgctgaa atgcggctgg tgcgcttcat ccgggcgaaa gaaccccgta ttggcaaata
     5341 ttgacggcca gttaagccat tcatgccagt aggcgcgcgg acgaaagtaa acccactggt
     5401 gataccattc gcgagcctcc ggatgacgac cgtagtgatg aatctctcct ggcgggaaca
     5461 gcaaaatatc acccggtcgg caaacaaatt ctcgtccctg atttttcacc accccctgac
     5521 cgcgaatggt gagattgaga atataacctt tcattcccag cggtcggtcg ataaaaaaat
     5581 cgagataacc gttggcctca atcggcgtta aacccgccac cagatgggca ttaaacgagt
     5641 atcccggcag caggggatca ttttgcgctt cagccatact tttcata
//
