LOCUS       sequence-389854-        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-Ca0.2
FEATURES             Location/Qualifiers
     source          1..5687
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        26..310
                     /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     233..252
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             356..373
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             377..409
                     /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(413..421,422..436)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     1090..1111
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1217..1238)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1478..1497)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2113..2130)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2283..2369
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2461..2488
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2507..2598
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2599..2667,2668..3459)
                     /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(2817..2836)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3630..4218
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4119..4138
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4372..4389
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4404..4544
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4470..4489)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4630..4652
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4808..5686)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFEISSVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 cttttcatac tcccgccatt cagagaagaa accaattgtc catattgcat cagacattgc
       61 cgtcactgcg tcttttactg gctcttctcg ctaaccaaac cggtaacccc gcttattaaa
      121 agcattctgt aacaaagcgg gaccaaagcc atgacaaaaa cgcgtaacaa aagtgtctat
      181 aatcacggca gaaaagtcca cattgattat ttgcacggcg tcacactttg ctatgccata
      241 gcatttttat ccataagatt agcggatcct acctgacgct ttttatcgca actctctact
      301 gtttctccat acccgttttt tgggctaaca ggaggaatta accatggggg gttctcatca
      361 tcatcatcat catggtatgg ctagcatgac tggtggacag caaatgggtc gggatctgta
      421 cgacgatgac gataaggatc cgagctcgag aatggcagaa atcggtactg gctttccatt
      481 cgacccccat tatgtggaag tcctgggcga gcgcatgcac tacgtcgatg ttggtccgcg
      541 cgatggcacc cctgtgctgt tcctgcacgg taacccgacc tcttcctacg tgtggcgcaa
      601 catcatcccg catgttgtac cgacccatcg ctgcattgct ccagacctga tcggtatggg
      661 caaatccgac aaaccagacc tgggttattt cttcgacgac cacgtccgct tcatggatgc
      721 cttcatcgaa gccctgggtc tggaagaggt cgtcctggtc attcacgact ggggctctgc
      781 tctgggtttc cactgggcca agcgcaatcc agagcgcgtc aaaggtattg catttatgga
      841 gttcatccgc cctatcccgt cctgggacga atggccaggt gaaaacgtct acatcaaggc
      901 cgacaagcag aagaacggca tcaaggcgga cttcaagatc cgccacgaca tcgaggacgg
      961 cggcgtgcag ctcgcctacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     1021 gctgcccgac aaccactacc tgagcgtcca gtccaaactt tcgaaagacc ccaacgagaa
     1081 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     1141 cgagctgtac aagggcggta ccggagggag catggtgagc aagggcgagg agctgttcac
     1201 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     1261 gtccggcgag ggtgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     1321 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
     1381 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     1441 cgaaggctac atccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     1501 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc gccgagctga agggcatcga
     1561 cttcaaggag gacggcaaca tcctggggca taagctggag tacaaccttg gcgcccgcgg
     1621 gaccttccag gccttccgca ccaccgacgt cggccgcaag ctgatcatcg atcagaacgt
     1681 ttttatcgag ggcacgctgc cgaagggtgt cgtccgcccg ctgactgaag tcgagatgga
     1741 ccattaccgc gagccgttcc tgaatcctgt tgaccgcgag ccactgtggc gcttcccaaa
     1801 cgagctgcca atcgccggtg agccagcgaa catcgtcgcg ctggtcgaag aatacatgga
     1861 ctggctgcac cagtcccctg tcccgaagct gctgttctgg ggcaccccag gcgttctgat
     1921 cccaccggcc gaagccgctc gcctggccaa aagcctgcct aactgcatgg ctgtggacat
     1981 cggcccgggt ctgaatctgc tgcaagaaga caacccggac ctgatcggca gcgagatcgc
     2041 gcgctggctg tcgacgcccg agatttccgg ctgaaagctt ggctgttttg gcggatgaga
     2101 gaagattttc agcctgatac agattaaatc agaacgcaga agcggtctga taaaacagaa
     2161 tttgcctggc ggcagtagcg cggtggtccc acctgacccc atgccgaact cagaagtgaa
     2221 acgccgtagc gccgatggta gtgtggggtc tccccatgcg agagtaggga actgccaggc
     2281 atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt
     2341 cggtgaacgc tctcctgagt aggacaaatc cgccgggagc ggatttgaac gttgcgaagc
     2401 aacggcccgg agggtggcgg gcaggacgcc cgccataaac tgccaggcat caaattaagc
     2461 agaaggccat cctgacggat ggcctttttg cgtttctaca aactcttttg tttatttttc
     2521 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     2581 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     2641 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     2701 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     2761 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     2821 tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac
     2881 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     2941 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     3001 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     3061 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     3121 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     3181 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     3241 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     3301 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     3361 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     3421 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     3481 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     3541 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     3601 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     3661 tgcttgcaaa caaaacaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     3721 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     3781 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     3841 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     3901 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     3961 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     4021 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     4081 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     4141 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     4201 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     4261 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     4321 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     4381 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     4441 atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     4501 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     4561 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     4621 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     4681 ggcagcagat caattcgcgc gcgaaggcga agcggcatgc ataatgtgcc tgtcaaatgg
     4741 acgaagcagg gattctgcaa accctatgct actccgtcaa gccgtcaatt gtctgattcg
     4801 ttaccaatta tgacaacttg acggctacat cattcacttt ttcttcacaa ccggcacgga
     4861 actcgctcgg gctggccccg gtgcattttt taaatacccg cgagaaatag agttgatcgt
     4921 caaaaccaac attgcgaccg acggtggcga taggcatccg ggtggtgctc aaaagcagct
     4981 tcgcctggct gatacgttgg tcctcgcgcc agcttaagac gctaatccct aactgctggc
     5041 ggaaaagatg tgacagacgc gacggcgaca agcaaacatg ctgtgcgacg ctggagattt
     5101 caaaattgct gtctgccagg tgatcgctga tgtactgaca agcctcgcgt acccgattat
     5161 ccatcggtgg atggagcgac tcgttaatcg cttccatgcg ccgcagtaac aattgctcaa
     5221 gcagatttat cgccagcagc tccgaatagc gcccttcccc ttgcccggcg ttaatgattt
     5281 gcccaaacag gtcgctgaaa tgcggctggt gcgcttcatc cgggcgaaag aaccccgtat
     5341 tggcaaatat tgacggccag ttaagccatt catgccagta ggcgcgcgga cgaaagtaaa
     5401 cccactggtg ataccattcg cgagcctccg gatgacgacc gtagtgatga atctctcctg
     5461 gcgggaacag caaaatatca cccggtcggc aaacaaattc tcgtccctga tttttcacca
     5521 ccccctgacc gcgaatggtg agattgagaa tataaccttt cattcccagc ggtcggtcga
     5581 taaaaaaatc gagataaccg ttggcctcaa tcggcgttaa acccgccacc agatgggcat
     5641 taaacgagta tcccggcagc aggggatcat tttgcgcttc agccata
//
