LOCUS       sequence-389856-        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-Ca1
FEATURES             Location/Qualifiers
     source          1..5687
                     /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     1085..1106
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1212..1233)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1473..1492)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2108..2125)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2278..2364
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2456..2483
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2502..2593
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2594..2662,2663..3454)
                     /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(2812..2831)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3625..4213
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4114..4133
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4367..4384
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4399..4539
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4465..4484)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4625..4647
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4803..5681)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFEISSVAQHVCLSPSRLSHLFRQQLGIS
                     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 tgcgccgacc 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 caggtgaaaa cgtctatatc aaggccgaca
      901 agcagaagaa cggcatcaag gcgaacttca agatccgcca caacatcgag gacggcggcg
      961 tgcaggctgc ctaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     1021 ccgacaacca cactttgagc gtccagtcca aactttcgaa agaccccaac gagaagcgcg
     1081 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     1141 tgtacaaggg cggtaccgga gggagcatgg tgagcaaggg cgaggagctg ttcaccgggg
     1201 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     1261 gcgagggtga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     1321 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     1381 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     1441 gctacatcca ggagcgcacc atcttcttcg gtgacgacgg caactacaag acccgcgccg
     1501 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     1561 aggaggacgg caacgtcctg gggcataagc tggcgtacaa ccatggcgct ggtggtacct
     1621 tccaggcctt ccgcaccacc gacgtcggcc gcgagctgat catcgatcag aacgttttta
     1681 tcgagggtac gctgccgatg ggtgtcgtcc gcccgctgac tgaagtcgag atggaccact
     1741 accgcgagcc gttcctgaat cctgttgacc gcgagccact gtggcgcttc ccaaacgagc
     1801 tgccaatcgc cggtgagcca gcgaacatcg tcgcgctggt cgaagaatac atggactggc
     1861 tgcaccagtc ccctgtcccg aagctgctgt tctggggcac cccaggcgtt ctgatcccac
     1921 cggccgaagc cgctcgcctg gccaaaagcc tgcctaactg caaggctgtg gacatcggcc
     1981 ctggtctgaa tctgctgcaa gaagacaacc cggacctgat cggcagcgag atcgcgcgct
     2041 ggctgtcgac gctggagatt tccggctgaa agcttggctg ttttggcgga tgagagaaga
     2101 ttttcagcct gatacagatt aaatcagaac gcagaagcgg tctgataaaa cagaatttgc
     2161 ctggcggcag tagcgcggtg gtcccacctg accccatgcc gaactcagaa gtgaaacgcc
     2221 gtagcgccga tggtagtgtg gggtctcccc atgcgagagt agggaactgc caggcatcaa
     2281 ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg
     2341 aacgctctcc tgagtaggac aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg
     2401 cccggagggt ggcgggcagg acgcccgcca taaactgcca ggcatcaaat taagcagaag
     2461 gccatcctga cggatggcct ttttgcgttt ctacaaactc ttttgtttat ttttctaaat
     2521 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     2581 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     2641 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     2701 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     2761 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     2821 cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc
     2881 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     2941 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     3001 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     3061 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     3121 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     3181 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     3241 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     3301 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     3361 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     3421 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     3481 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     3541 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     3601 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     3661 gcaaacaaaa caaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     3721 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     3781 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     3841 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     3901 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     3961 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     4021 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     4081 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     4141 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     4201 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     4261 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     4321 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     4381 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     4441 acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     4501 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
     4561 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     4621 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
     4681 cagatcaatt cgcgcgcgaa ggcgaagcgg catgcataat gtgcctgtca aatggacgaa
     4741 gcagggattc tgcaaaccct atgctactcc gtcaagccgt caattgtctg attcgttacc
     4801 aattatgaca acttgacggc tacatcattc actttttctt cacaaccggc acggaactcg
     4861 ctcgggctgg ccccggtgca ttttttaaat acccgcgaga aatagagttg atcgtcaaaa
     4921 ccaacattgc gaccgacggt ggcgataggc atccgggtgg tgctcaaaag cagcttcgcc
     4981 tggctgatac gttggtcctc gcgccagctt aagacgctaa tccctaactg ctggcggaaa
     5041 agatgtgaca gacgcgacgg cgacaagcaa acatgctgtg cgacgctgga gatttcaaaa
     5101 ttgctgtctg ccaggtgatc gctgatgtac tgacaagcct cgcgtacccg attatccatc
     5161 ggtggatgga gcgactcgtt aatcgcttcc atgcgccgca gtaacaattg ctcaagcaga
     5221 tttatcgcca gcagctccga atagcgccct tccccttgcc cggcgttaat gatttgccca
     5281 aacaggtcgc tgaaatgcgg ctggtgcgct tcatccgggc gaaagaaccc cgtattggca
     5341 aatattgacg gccagttaag ccattcatgc cagtaggcgc gcggacgaaa gtaaacccac
     5401 tggtgatacc attcgcgagc ctccggatga cgaccgtagt gatgaatctc tcctggcggg
     5461 aacagcaaaa tatcacccgg tcggcaaaca aattctcgtc cctgattttt caccaccccc
     5521 tgaccgcgaa tggtgagatt gagaatataa cctttcattc ccagcggtcg gtcgataaaa
     5581 aaatcgagat aaccgttggc ctcaatcggc gttaaacccg ccaccagatg ggcattaaac
     5641 gagtatcccg gcagcagggg atcattttgc gcttcagcca tactttt
//
