LOCUS       sequence-389889-        5651 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 5651)
  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 5651)
  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-165-178
FEATURES             Location/Qualifiers
     source          1..5651
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        33..317
                     /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     240..259
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             363..380
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             384..416
                     /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(420..428,429..443)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     1163..1184
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1290..1311)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1551..1570)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2084..2101)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2254..2340
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2432..2459
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2478..2569
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2570..2638,2639..3430)
                     /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(2788..2807)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3601..4189
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4090..4109
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4343..4360
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4375..4515
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4441..4460)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4601..4623
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(join(4779..5651,1..6))
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 agccatactt ttcatactcc cgccattcag agaagaaacc aattgtccat attgcatcag
       61 acattgccgt cactgcgtct tttactggct cttctcgcta accaaaccgg taaccccgct
      121 tattaaaagc attctgtaac aaagcgggac caaagccatg acaaaaacgc gtaacaaaag
      181 tgtctataat cacggcagaa aagtccacat tgattatttg cacggcgtca cactttgcta
      241 tgccatagca tttttatcca taagattagc ggatcctacc tgacgctttt tatcgcaact
      301 ctctactgtt tctccatacc cgttttttgg gctaacagga ggaattaacc atggggggtt
      361 ctcatcatca tcatcatcat ggtatggcta gcatgactgg tggacagcaa atgggtcggg
      421 atctgtacga cgatgacgat aaggatccga gctcgagaat ggcagaaatc ggtactggct
      481 ttccattcga cccccattat gtggaagtcc tgggcgagcg catgcactac gtcgatgttg
      541 gtccgcgcga tggcacccct gtgctgttcc tgcacggtaa cccgacctcc tcctacgtgt
      601 ggcgcaacat catcccgcat gttgcaccga cccatcgctg cattgctcca gacctgatcg
      661 gtatgggcaa atccgacaaa ccagacctgg gttatttctt cgacgaccac gtccgcttca
      721 tggatgcctt catcgaagcc ctgggtctgg aagaggtcgt cctggtcatt cacgactggg
      781 gctccgctct gggtttccac tgggccaagc gcaatccaga gcgcgtcaaa ggtattgcat
      841 ttatggagtt catccgccct atcccgacct gggacgaatg gccagaattt gcccgcgaga
      901 ccttccaggc cttccgcacc accgacgtcg gccgcaagct gatcatcgat ggtgaaaacg
      961 tctatatcaa ggccgacaag cagaagaacg gcatcaaggc gaacttcaag atccgccaca
     1021 acatcgagga cggcggcgtg cagctcgcct accactacca gcagaacacc cccatcggcg
     1081 acggccccgt gctgctgccc gacaaccact acctgagcgt ccagtccaaa ctttcgaaag
     1141 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     1201 ctctcggcat ggacgagctg tacaagggcg gtaccggagg gagcatggtg agcaagggcg
     1261 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
     1321 acaagttcag cgtgtccggc gagggtgagg gcgatgccac ctacggcaag ctgaccctga
     1381 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga
     1441 cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca
     1501 agtccgccat gcccgaaggc tacatccagg agcgcaccat cttcttcaag gacgacggca
     1561 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc
     1621 tgaagggcat cgacttcaag gaggacggca acatcctggg gcataagctg gagtacaacc
     1681 ttggccgccc gctgactgaa gtcgagatgg accattaccg cgagccgttc ctgaatcctg
     1741 ttgaccgcga gccactgtgg cgcttcccaa acgagctgcc aatcgccggt gagccagcga
     1801 acatcgtcgc gctggtcgaa gaatacatgg actggctgca ccagtcccct gtcccgaagc
     1861 tgctgttctg gggcacccca ggcgttctga tcccaccggc cgaagccgct cgcctggcca
     1921 aaagcctgcc taactgcaag gctgtggaca tcggcccggg tctgaatctg ctgcaagaag
     1981 acaacccgga cctgatcggc agcgagatcg cgcgctggct gtcgacgctc gagatttccg
     2041 gctgaaagct tggctgtttt ggcggatgag agaagatttt cagcctgata cagattaaat
     2101 cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc
     2161 cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt
     2221 ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa
     2281 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat
     2341 ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg ggcaggacgc
     2401 ccgccataaa ctgccaggca tcaaattaag cagaaggcca tcctgacgga tggccttttt
     2461 gcgtttctac aaactctttt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     2521 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     2581 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     2641 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     2701 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     2761 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc
     2821 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     2881 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     2941 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     3001 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     3061 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     3121 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     3181 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     3241 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     3301 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     3361 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     3421 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     3481 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     3541 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     3601 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaacaac caccgctacc
     3661 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     3721 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     3781 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     3841 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     3901 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     3961 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     4021 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     4081 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     4141 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     4201 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     4261 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     4321 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     4381 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag
     4441 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     4501 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     4561 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     4621 aggttttcac cgtcatcacc gaaacgcgcg aggcagcaga tcaattcgcg cgcgaaggcg
     4681 aagcggcatg cataatgtgc ctgtcaaatg gacgaagcag ggattctgca aaccctatgc
     4741 tactccgtca agccgtcaat tgtctgattc gttaccaatt atgacaactt gacggctaca
     4801 tcattcactt tttcttcaca accggcacgg aactcgctcg ggctggcccc ggtgcatttt
     4861 ttaaataccc gcgagaaata gagttgatcg tcaaaaccaa cattgcgacc gacggtggcg
     4921 ataggcatcc gggtggtgct caaaagcagc ttcgcctggc tgatacgttg gtcctcgcgc
     4981 cagcttaaga cgctaatccc taactgctgg cggaaaagat gtgacagacg cgacggcgac
     5041 aagcaaacat gctgtgcgac gctggcgata tcaaaattgc tgtctgccag gtgatcgctg
     5101 atgtactgac aagcctcgcg tacccgatta tccatcggtg gatggagcga ctcgttaatc
     5161 gcttccatgc gccgcagtaa caattgctca agcagattta tcgccagcag ctccgaatag
     5221 cgcccttccc cttgcccggc gttaatgatt tgcccaaaca ggtcgctgaa atgcggctgg
     5281 tgcgcttcat ccgggcgaaa gaaccccgta ttggcaaata ttgacggcca gttaagccat
     5341 tcatgccagt aggcgcgcgg acgaaagtaa acccactggt gataccattc gcgagcctcc
     5401 ggatgacgac cgtagtgatg aatctctcct ggcgggaaca gcaaaatatc acccggtcgg
     5461 caaacaaatt ctcgtccctg atttttcacc accccctgac cgcgaatggt gagattgaga
     5521 atataacctt tcattcccag cggtcggtcg ataaaaaaat cgagataacc gttggcctca
     5581 atcggcgtta aacccgccac cagatgggca ttaaacgagt atcccggcag caggggatca
     5641 ttttgcgctt c
//
