LOCUS       sequence-389885-        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-159-162
FEATURES             Location/Qualifiers
     source          1..5681
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        21..305
                     /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     228..247
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             351..368
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             372..404
                     /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(408..416,417..431)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     1133..1154
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1260..1281)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1521..1540)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2102..2119)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2272..2358
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2450..2477
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2496..2587
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2588..2656,2657..3448)
                     /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(2806..2825)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3619..4207
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4108..4127
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4361..4378
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4393..4533
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4459..4478)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4619..4641
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4797..5675)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 catactcccg ccattcagag aagaaaccaa ttgtccatat tgcatcagac attgccgtca
       61 ctgcgtcttt tactggctct tctcgctaac caaaccggta accccgctta ttaaaagcat
      121 tctgtaacaa agcgggacca aagccatgac aaaaacgcgt aacaaaagtg tctataatca
      181 cggcagaaaa gtccacattg attatttgca cggcgtcaca ctttgctatg ccatagcatt
      241 tttatccata agattagcgg atcctacctg acgcttttta tcgcaactct ctactgtttc
      301 tccatacccg ttttttgggc taacaggagg aattaaccat ggggggttct catcatcatc
      361 atcatcatgg tatggctagc atgactggtg gacagcaaat gggtcgggat ctgtacgacg
      421 atgacgataa ggatccgagc tcgagaatgg cagaaatcgg tactggcttt ccattcgacc
      481 cccattatgt ggaagtcctg ggcgagcgca tgcactacgt cgatgttggt ccgcgcgatg
      541 gcacccctgt gctgttcctg cacggtaacc cgacctcctc ctacgtgtgg cgcaacatca
      601 tcccgcatgt tgcaccgacc catcgctgca ttgctccaga cctgatcggt atgggcaaat
      661 ccgacaaacc agacctgggt tatttcttcg acgaccacgt ccgcttcatg gatgccttca
      721 tcgaagccct gggtctggaa gaggtcgtcc tggtcattca cgactggggc tccgctctgg
      781 gtttccactg ggccaagcgc aatccagagc gcgtcaaagg tattgcattt atggagttca
      841 tccgccctat cccgacctgg gacgaatggc cagaatttgc ccgcgagacc ttccaggcct
      901 tccgcaccac cgacgtcggc ggtgaaaacg tctatatcaa ggccgacaag cagaagaacg
      961 gcatcaaggc gaacttcaag atccgccaca acatcgagga cggcggcgtg cagctcgcct
     1021 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     1081 acctgagcgt ccagtccaaa ctttcgaaag accccaacga gaagcgcgat cacatggtcc
     1141 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagggcg
     1201 gtaccggagg gagcatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     1261 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggtgagg
     1321 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     1381 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
     1441 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacatccagg
     1501 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     1561 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     1621 acatcctggg gcataagctg gagtacaacc ttggcatcga tcagaacgtt tttatcgagg
     1681 gtacgctgcc gatgggtgtc gtccgcccgc tgactgaagt cgagatggac cattaccgcg
     1741 agccgttcct gaatcctgtt gaccgcgagc cactgtggcg cttcccaaac gagctgccaa
     1801 tcgccggtga gccagcgaac atcgtcgcgc tggtcgaaga atacatggac tggctgcacc
     1861 agtcccctgt cccgaagctg ctgttctggg gcaccccagg cgttctgatc ccaccggccg
     1921 aagccgctcg cctggccaaa agcctgccta actgcaaggc tgtggacatc ggcccgggtc
     1981 tgaatctgct gcaagaagac aacccggacc tgatcggcag cgagatcgcg cgctggctgt
     2041 cgacgctcga gatttccggc tgaaagcttg gctgttttgg cggatgagag aagattttca
     2101 gcctgataca gattaaatca gaacgcagaa gcggtctgat aaaacagaat ttgcctggcg
     2161 gcagtagcgc ggtggtccca cctgacccca tgccgaactc agaagtgaaa cgccgtagcg
     2221 ccgatggtag tgtggggtct ccccatgcga gagtagggaa ctgccaggca tcaaataaaa
     2281 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct
     2341 ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca acggcccgga
     2401 gggtggcggg caggacgccc gccataaact gccaggcatc aaattaagca gaaggccatc
     2461 ctgacggatg gcctttttgc gtttctacaa actcttttgt ttatttttct aaatacattc
     2521 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     2581 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     2641 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     2701 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     2761 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     2821 attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     2881 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     2941 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     3001 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     3061 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     3121 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     3181 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     3241 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     3301 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     3361 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     3421 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     3481 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     3541 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     3601 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     3661 aaaacaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     3721 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     3781 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     3841 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     3901 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     3961 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     4021 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     4081 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     4141 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     4201 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     4261 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     4321 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     4381 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     4441 catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     4501 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     4561 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     4621 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagcagatc
     4681 aattcgcgcg cgaaggcgaa gcggcatgca taatgtgcct gtcaaatgga cgaagcaggg
     4741 attctgcaaa ccctatgcta ctccgtcaag ccgtcaattg tctgattcgt taccaattat
     4801 gacaacttga cggctacatc attcactttt tcttcacaac cggcacggaa ctcgctcggg
     4861 ctggccccgg tgcatttttt aaatacccgc gagaaataga gttgatcgtc aaaaccaaca
     4921 ttgcgaccga cggtggcgat aggcatccgg gtggtgctca aaagcagctt cgcctggctg
     4981 atacgttggt cctcgcgcca gcttaagacg ctaatcccta actgctggcg gaaaagatgt
     5041 gacagacgcg acggcgacaa gcaaacatgc tgtgcgacgc tggcgatatc aaaattgctg
     5101 tctgccaggt gatcgctgat gtactgacaa gcctcgcgta cccgattatc catcggtgga
     5161 tggagcgact cgttaatcgc ttccatgcgc cgcagtaaca attgctcaag cagatttatc
     5221 gccagcagct ccgaatagcg cccttcccct tgcccggcgt taatgatttg cccaaacagg
     5281 tcgctgaaat gcggctggtg cgcttcatcc gggcgaaaga accccgtatt ggcaaatatt
     5341 gacggccagt taagccattc atgccagtag gcgcgcggac gaaagtaaac ccactggtga
     5401 taccattcgc gagcctccgg atgacgaccg tagtgatgaa tctctcctgg cgggaacagc
     5461 aaaatatcac ccggtcggca aacaaattct cgtccctgat ttttcaccac cccctgaccg
     5521 cgaatggtga gattgagaat ataacctttc attcccagcg gtcggtcgat aaaaaaatcg
     5581 agataaccgt tggcctcaat cggcgttaaa cccgccacca gatgggcatt aaacgagtat
     5641 cccggcagca ggggatcatt ttgcgcttca gccatacttt t
//
