LOCUS       sequence-389875-        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-173-174
FEATURES             Location/Qualifiers
     source          1..5687
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        32..316
                     /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     239..258
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             362..379
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             383..415
                     /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(419..427,428..442)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     1186..1207
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1313..1334)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1574..1593)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2119..2136)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2289..2375
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2467..2494
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2513..2604
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2605..2673,2674..3465)
                     /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(2823..2842)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3636..4224
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4125..4144
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4378..4395
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4410..4550
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4476..4495)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4636..4658
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(join(4814..5687,1..5))
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 gccatacttt tcatactccc gccattcaga gaagaaacca attgtccata ttgcatcaga
       61 cattgccgtc actgcgtctt ttactggctc ttctcgctaa ccaaaccggt aaccccgctt
      121 attaaaagca ttctgtaaca aagcgggacc aaagccatga caaaaacgcg taacaaaagt
      181 gtctataatc acggcagaaa agtccacatt gattatttgc acggcgtcac actttgctat
      241 gccatagcat ttttatccat aagattagcg gatcctacct gacgcttttt atcgcaactc
      301 tctactgttt ctccataccc gttttttggg ctaacaggag gaattaacca tggggggttc
      361 tcatcatcat catcatcatg gtatggctag catgactggt ggacagcaaa tgggtcggga
      421 tctgtacgac gatgacgata aggatccgag ctcgagaatg gcagaaatcg gtactggctt
      481 tccattcgac ccccattatg tggaagtcct gggcgagcgc atgcactacg tcgatgttgg
      541 tccgcgcgat ggcacccctg tgctgttcct gcacggtaac ccgacctcct cctacgtgtg
      601 gcgcaacatc atcccgcatg ttgcaccgac ccatcgctgc attgctccag acctgatcgg
      661 tatgggcaaa tccgacaaac cagacctggg ttatttcttc gacgaccacg tccgcttcat
      721 ggatgccttc atcgaagccc tgggtctgga agaggtcgtc ctggtcattc acgactgggg
      781 ctccgctctg ggtttccact gggccaagcg caatccagag cgcgtcaaag gtattgcatt
      841 tatggagttc atccgcccta tcccgacctg ggacgaatgg ccagaatttg cccgcgagac
      901 cttccaggcc ttccgcacca ccgacgtcgg ccgcaagctg atcatcgatc agaacgtttt
      961 tatcgagggt acgggtgaaa acgtctatat caaggccgac aagcagaaga acggcatcaa
     1021 ggcgaacttc aagatccgcc acaacatcga ggacggcggc gtgcagctcg cctaccacta
     1081 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     1141 cgtccagtcc aaactttcga aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     1201 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagg gcggtaccgg
     1261 agggagcatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
     1321 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggtg agggcgatgc
     1381 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     1441 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca
     1501 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacatcc aggagcgcac
     1561 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     1621 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     1681 ggggcataag ctggagtaca accttggcat gggtgtcgtc cgcccgctga ctgaagtcga
     1741 gatggaccat taccgcgagc cgttcctgaa tcctgttgac cgcgagccac tgtggcgctt
     1801 cccaaacgag ctgccaatcg ccggtgagcc agcgaacatc gtcgcgctgg tcgaagaata
     1861 catggactgg ctgcaccagt cccctgtccc gaagctgctg ttctggggca ccccaggcgt
     1921 tctgatccca ccggccgaag ccgctcgcct ggccaaaagc ctgcctaact gcaaggctgt
     1981 ggacatcggc ccgggtctga atctgctgca agaagacaac ccggacctga tcggcagcga
     2041 gatcgcgcgc tggctgtcga cgctcgagat ttccggctga aagcttggct gttttggcgg
     2101 atgagagaag attttcagcc tgatacagat taaatcagaa cgcagaagcg gtctgataaa
     2161 acagaatttg cctggcggca gtagcgcggt ggtcccacct gaccccatgc cgaactcaga
     2221 agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc catgcgagag tagggaactg
     2281 ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     2341 gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat ttgaacgttg
     2401 cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa
     2461 ttaagcagaa ggccatcctg acggatggcc tttttgcgtt tctacaaact cttttgttta
     2521 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     2581 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     2641 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     2701 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     2761 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     2821 ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc aagagcaact cggtcgccgc
     2881 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     2941 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     3001 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     3061 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     3121 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     3181 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     3241 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     3301 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     3361 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     3421 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     3481 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     3541 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     3601 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     3661 atctgctgct tgcaaacaaa acaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     3721 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     3781 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     3841 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     3901 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     3961 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     4021 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     4081 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     4141 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     4201 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     4261 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     4321 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     4381 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     4441 tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag
     4501 ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc
     4561 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     4621 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     4681 gcgcgaggca gcagatcaat tcgcgcgcga aggcgaagcg gcatgcataa tgtgcctgtc
     4741 aaatggacga agcagggatt ctgcaaaccc tatgctactc cgtcaagccg tcaattgtct
     4801 gattcgttac caattatgac aacttgacgg ctacatcatt cactttttct tcacaaccgg
     4861 cacggaactc gctcgggctg gccccggtgc attttttaaa tacccgcgag aaatagagtt
     4921 gatcgtcaaa accaacattg cgaccgacgg tggcgatagg catccgggtg gtgctcaaaa
     4981 gcagcttcgc ctggctgata cgttggtcct cgcgccagct taagacgcta atccctaact
     5041 gctggcggaa aagatgtgac agacgcgacg gcgacaagca aacatgctgt gcgacgctgg
     5101 cgatatcaaa attgctgtct gccaggtgat cgctgatgta ctgacaagcc tcgcgtaccc
     5161 gattatccat cggtggatgg agcgactcgt taatcgcttc catgcgccgc agtaacaatt
     5221 gctcaagcag atttatcgcc agcagctccg aatagcgccc ttccccttgc ccggcgttaa
     5281 tgatttgccc aaacaggtcg ctgaaatgcg gctggtgcgc ttcatccggg cgaaagaacc
     5341 ccgtattggc aaatattgac ggccagttaa gccattcatg ccagtaggcg cgcggacgaa
     5401 agtaaaccca ctggtgatac cattcgcgag cctccggatg acgaccgtag tgatgaatct
     5461 ctcctggcgg gaacagcaaa atatcacccg gtcggcaaac aaattctcgt ccctgatttt
     5521 tcaccacccc ctgaccgcga atggtgagat tgagaatata acctttcatt cccagcggtc
     5581 ggtcgataaa aaaatcgaga taaccgttgg cctcaatcgg cgttaaaccc gccaccagat
     5641 gggcattaaa cgagtatccc ggcagcaggg gatcattttg cgcttca
//
