LOCUS       sequence-384163-        7637 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Far-red fluorescent Ca2+ ion indicator for mammalian cell and 
            bacterial expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7637)
  AUTHORS   Hashizume R, Fujii H, Mehta S, Ota K, Qian Y, Zhu W, Drobizhev M, 
            Nasu Y, Zhang J, Bito H, Campbell RE
  TITLE     A genetically encoded far-red fluorescent calcium ion biosensor 
            derived from a biliverdin-binding protein.
  JOURNAL   Protein Sci. 2022 Oct;31(10):e4440. doi: 10.1002/pro.4440.
  PUBMED    36173169
REFERENCE   2  (bases 1 to 7637)
  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"; journalName: "Protein 
            Sci."; date: "2022-10"; volume: "31(10):e4440. doi"; pages: " 
            10.1002/pro.4440"
COMMENT     Sequence Label: pDuex2-iBB-GECO1
FEATURES             Location/Qualifiers
     source          1..7637
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        81..460
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        461..664
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     615..635
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             2278..2295
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3074..3091)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3080..3304
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3350..3778
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3437..3456)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3647..3668
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3787..3807)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3792..4121
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3972..4107
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4034..4053
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4188..4982
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4242..4261)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4852..4871
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5156..5277
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5193..5212)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5247..5266
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5326..5342)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5326..5342)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5339..5361)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5350..5366
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5374..5380,5381..5398,5399..5404))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5419..5440
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5557..5574)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5728..6316)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5808..5827)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6487..7278,7279..7347))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7110..7129
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7348..7452)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7527..7546)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt
       61 acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac
      121 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg
      181 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
      241 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
      301 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
      361 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
      421 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta
      481 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga
      541 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa
      601 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag
      661 agctctctgg ctaactagag aacccactgc ttactggtcc ggattcaatt aatcatcggc
      721 tcgtataata tacccgtttt tttgggctag aaataatttt gtctaacttt aagaaggaga
      781 tatctcgaga tgttctcggt accgctgact acctcagctt tcggccacgc gtttctggct
      841 aacattgaac gtgagcagat ccacctggcg ggctccattc agccgcacgg tatcctgctg
      901 gctgttaaag agcctgacaa cgtggtgatc caggcttcta ttaacgctgc ggagttcctg
      961 aacaccaact ttgttgttgg ccgtccgctg cgtgacctgg gcggcgatct gcctttgcag
     1021 atcctgccgc acctgaacgg cccgctgcac ctggctccga tgaccctgcg ttgtactgtg
     1081 ggttctccgc cgcgtcgtgt ggactgtacc attcatcgac cgtctaacgg cggcctgatc
     1141 gtagaactgg aaccagcaac caaggccact aacattgcgc cggctctggt cggtgcgctt
     1201 catcgtatca cttcttcatc ctccctgatg ggcctgtgtg acgaaaccgc gactattttc
     1261 cgtgagatca ctggtttcga ccgtgtgatg gtaatgcgtc tcggcgcgct tgacgatctg
     1321 actgaagagc agatcgcaga gattaaagag gctttctccc tatttgacaa ggacggggac
     1381 gggacgataa caaccaagga gctggggacg gtgttccggt ctctggggca gaaccccaca
     1441 gaagcagagc tgcaggactt aatcaacgag gtagatgccg atggcaacgg catattcgac
     1501 ttccctgagt tcctgacgat gatggcaagg aaaatgaatg acgcagacag tgaagaggaa
     1561 attagaggag cgttccgcgt gtttgataag gacggcaatg gcaacatcgg cgcagcaggg
     1621 cttcgccacg tgatgtcaga ccttggtgag aagttaactg atgaggaggt tgatgaaatg
     1681 atcagggtag cggacaacga tggggatggt caggtaaact acgaagagtt tgtacaaatc
     1741 atgacagcga agggaggttc tgtagattca tcacgtcgta agtggaataa ggcaggacac
     1801 gcagtcagag ctataggtcg gctgagctcg cgtaggcatg atttgctttc cgaatgtcgt
     1861 cgtgcggacc tggaagcgtt cctgggtaac cgctacccgg cgtatactat tccgcagacc
     1921 gctcgtcgcc tgtacgaact caaccgtgtt cgcctgatgg tagatgtgaa ctatactccg
     1981 gttcctctag agccgcgcat cagcccgctg aacggtcgtg atctggatat gtccctgtct
     2041 tgcctgcgct ctatgtcccc gtgccacctg aaatacatgc aggacatggg cgtgggcgca
     2101 accctggttt gctctctgat ggtgtctggt cgtctgtggg gtctgatcgt ttgccaccac
     2161 tacgaaccgc gctacgttcc gtcccacatt cgcgctgctg gcgaagcgct ggcggaagca
     2221 tgtgcgaacc gcatcgcgac gctggagagc tttgcaccgt ctcagtccaa aaagcttcat
     2281 catcaccatc accattgaaa gaaggagata tacataccca tgagtgtcaa cttagcttcc
     2341 cagttgcggg aagggacgaa aaaatcccac tccatggcgg agaacgtcgg ctttgtcaaa
     2401 tgcttcctca agggcgttgt cgagaaaaat tcctaccgta agctggttgg caatctctac
     2461 tttgtctaca gtgccatgga agaggaaatg gcaaaattta aggaccatcc catcctcagc
     2521 cacatttact tccccgaact caaccgcaaa caaagcctag agcaagacct gcaattctat
     2581 tacggctcca actggcggca agaagtgaaa atttctgccg ctggccaagc ctatgtggac
     2641 cgagtccggc aagtggccgc tacggcccct gaattgttgg tggcccattc ctacacccgt
     2701 tacctggggg atctttccgg cggtcaaatt ctcaagaaaa ttgcccaaaa tgccatgaat
     2761 ctccacgatg gtggcacagc tttctatgaa tttgccgaca ttgatgacga aaaggctttt
     2821 aaaaatacct accgtcaagc tatgaatgat ctgcccattg accaagccac cgccgaacgg
     2881 attgtggatg aagccaatga cgcctttgcc atgaacatga aaatgttcaa cgaacttgaa
     2941 ggcaacctga tcaaggcgat cggcattatg gtgttcaaca gcctcacccg tcgccgcagt
     3001 caaggcagca ccgaagttgg cctcgccacc tccgaaggct aatctagagg gcccgtttaa
     3061 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     3121 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     3181 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     3241 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     3301 atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt
     3361 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     3421 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     3481 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     3541 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     3601 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     3661 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     3721 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa
     3781 ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3841 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     3901 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     3961 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     4021 gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga
     4081 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta
     4141 tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag
     4201 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     4261 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     4321 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
     4381 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     4441 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     4501 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     4561 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     4621 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     4681 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg
     4741 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     4801 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     4861 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     4921 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     4981 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga
     5041 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     5101 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
     5161 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     5221 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     5281 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     5341 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     5401 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     5461 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     5521 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     5581 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     5641 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     5701 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     5761 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     5821 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     5881 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     5941 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     6001 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     6061 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     6121 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     6181 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     6241 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     6301 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     6361 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     6421 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     6481 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     6541 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     6601 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     6661 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     6721 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     6781 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     6841 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     6901 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     6961 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     7021 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     7081 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     7141 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     7201 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     7261 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     7321 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     7381 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     7441 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct
     7501 cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     7561 tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct
     7621 acaacaaggc aaggctt
//
