LOCUS       sequence-389869-        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-147-150
FEATURES             Location/Qualifiers
     source          1..5681
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        24..308
                     /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     231..250
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             354..371
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             375..407
                     /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(411..419,420..434)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     1100..1121
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1227..1248)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1488..1507)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2105..2122)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2275..2361
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2453..2480
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2499..2590
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2591..2659,2660..3451)
                     /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(2809..2828)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3622..4210
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4111..4130
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4364..4381
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4396..4536
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4462..4481)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4622..4644
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4800..5678)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 tttcatactc ccgccattca gagaagaaac caattgtcca tattgcatca gacattgccg
       61 tcactgcgtc ttttactggc tcttctcgct aaccaaaccg gtaaccccgc ttattaaaag
      121 cattctgtaa caaagcggga ccaaagccat gacaaaaacg cgtaacaaaa gtgtctataa
      181 tcacggcaga aaagtccaca ttgattattt gcacggcgtc acactttgct atgccatagc
      241 atttttatcc ataagattag cggatcctac ctgacgcttt ttatcgcaac tctctactgt
      301 ttctccatac ccgttttttg ggctaacagg aggaattaac catggggggt tctcatcatc
      361 atcatcatca tggtatggct agcatgactg gtggacagca aatgggtcgg gatctgtacg
      421 acgatgacga taaggatccg agctcgagaa tggcagaaat cggtactggc tttccattcg
      481 acccccatta tgtggaagtc ctgggcgagc gcatgcacta cgtcgatgtt ggtccgcgcg
      541 atggcacccc tgtgctgttc ctgcacggta acccgacctc ctcctacgtg tggcgcaaca
      601 tcatcccgca tgttgcaccg acccatcgct gcattgctcc agacctgatc ggtatgggca
      661 aatccgacaa accagacctg ggttatttct tcgacgacca cgtccgcttc atggatgcct
      721 tcatcgaagc cctgggtctg gaagaggtcg tcctggtcat tcacgactgg ggctccgctc
      781 tgggtttcca ctgggccaag cgcaatccag agcgcgtcaa aggtattgca tttatggagt
      841 tcatccgccc tatcccgacc tgggacgaat ggccagaatt tgcccgcggt gaaaacgtct
      901 atatcaaggc cgacaagcag aagaacggca tcaaggcgaa cttcaagatc cgccacaaca
      961 tcgaggacgg cggcgtgcag ctcgcctacc actaccagca gaacaccccc atcggcgacg
     1021 gccccgtgct gctgcccgac aaccactacc tgagcgtcca gtccaaactt tcgaaagacc
     1081 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     1141 tcggcatgga cgagctgtac aagggcggta ccggagggag catggtgagc aagggcgagg
     1201 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     1261 agttcagcgt gtccggcgag ggtgagggcg atgccaccta cggcaagctg accctgaagt
     1321 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     1381 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     1441 ccgccatgcc cgaaggctac atccaggagc gcaccatctt cttcaaggac gacggcaact
     1501 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     1561 agggcatcga cttcaaggag gacggcaaca tcctggggca taagctggag tacaaccttg
     1621 gcgccttccg caccaccgac gtcggccgca agctgatcat cgatcagaac gtttttatcg
     1681 agggtacgct gccgatgggt gtcgtccgcc cgctgactga agtcgagatg gaccattacc
     1741 gcgagccgtt cctgaatcct gttgaccgcg agccactgtg gcgcttccca aacgagctgc
     1801 caatcgccgg tgagccagcg aacatcgtcg cgctggtcga agaatacatg gactggctgc
     1861 accagtcccc tgtcccgaag ctgctgttct ggggcacccc aggcgttctg atcccaccgg
     1921 ccgaagccgc tcgcctggcc aaaagcctgc ctaactgcaa ggctgtggac atcggcccgg
     1981 gtctgaatct gctgcaagaa gacaacccgg acctgatcgg cagcgagatc gcgcgctggc
     2041 tgtcgacgct cgagatttcc ggctgaaagc ttggctgttt tggcggatga gagaagattt
     2101 tcagcctgat acagattaaa tcagaacgca gaagcggtct gataaaacag aatttgcctg
     2161 gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta
     2221 gcgccgatgg tagtgtgggg tctccccatg cgagagtagg gaactgccag gcatcaaata
     2281 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
     2341 gctctcctga gtaggacaaa tccgccggga gcggatttga acgttgcgaa gcaacggccc
     2401 ggagggtggc gggcaggacg cccgccataa actgccaggc atcaaattaa gcagaaggcc
     2461 atcctgacgg atggcctttt tgcgtttcta caaactcttt tgtttatttt tctaaataca
     2521 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     2581 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     2641 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     2701 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     2761 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     2821 ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     2881 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     2941 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     3001 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     3061 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     3121 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     3181 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     3241 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     3301 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     3361 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     3421 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     3481 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     3541 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     3601 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3661 aacaaaacaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3721 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     3781 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     3841 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     3901 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     3961 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     4021 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     4081 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     4141 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     4201 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4261 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     4321 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     4381 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     4441 ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     4501 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     4561 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     4621 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagcag
     4681 atcaattcgc gcgcgaaggc gaagcggcat gcataatgtg cctgtcaaat ggacgaagca
     4741 gggattctgc aaaccctatg ctactccgtc aagccgtcaa ttgtctgatt cgttaccaat
     4801 tatgacaact tgacggctac atcattcact ttttcttcac aaccggcacg gaactcgctc
     4861 gggctggccc cggtgcattt tttaaatacc cgcgagaaat agagttgatc gtcaaaacca
     4921 acattgcgac cgacggtggc gataggcatc cgggtggtgc tcaaaagcag cttcgcctgg
     4981 ctgatacgtt ggtcctcgcg ccagcttaag acgctaatcc ctaactgctg gcggaaaaga
     5041 tgtgacagac gcgacggcga caagcaaaca tgctgtgcga cgctggcgat atcaaaattg
     5101 ctgtctgcca ggtgatcgct gatgtactga caagcctcgc gtacccgatt atccatcggt
     5161 ggatggagcg actcgttaat cgcttccatg cgccgcagta acaattgctc aagcagattt
     5221 atcgccagca gctccgaata gcgcccttcc ccttgcccgg cgttaatgat ttgcccaaac
     5281 aggtcgctga aatgcggctg gtgcgcttca tccgggcgaa agaaccccgt attggcaaat
     5341 attgacggcc agttaagcca ttcatgccag taggcgcgcg gacgaaagta aacccactgg
     5401 tgataccatt cgcgagcctc cggatgacga ccgtagtgat gaatctctcc tggcgggaac
     5461 agcaaaatat cacccggtcg gcaaacaaat tctcgtccct gatttttcac caccccctga
     5521 ccgcgaatgg tgagattgag aatataacct ttcattccca gcggtcggtc gataaaaaaa
     5581 tcgagataac cgttggcctc aatcggcgtt aaacccgcca ccagatgggc attaaacgag
     5641 tatcccggca gcaggggatc attttgcgct tcagccatac t
//
