LOCUS       sequence-230227-        6734 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Negative-control mutant for sapphireAKAR biosensor..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6734)
  AUTHORS   Mehta S, Zhang Y, Roth RH, Zhang JF, Mo A, Tenner B, Huganir RL, 
            Zhang J
  TITLE     Single-fluorophore biosensors for sensitive and multiplexed 
            detection of signalling activities.
  JOURNAL   Nat Cell Biol. 2018 Sep 24. pii: 10.1038/s41556-018-0200-6. doi: 
            10.1038/s41556-018-0200-6.
  PUBMED    30250062
REFERENCE   2  (bases 1 to 6734)
  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: "Nat Cell 
            Biol. 2018 Sep 24. pii: 10.1038/s41556-018-0200-6. doi: 
            10.1038/s41556-018-0200-6."
COMMENT     Sequence Label: pcDNA3.1(+)-sapphireAKAR(T/A)
FEATURES             Location/Qualifiers
     source          1..6734
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             922..939
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             943..975
                     /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(979..987,988..1002)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     promoter        complement(2287..2305)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2288..2305)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2325..2342)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2331..2555
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2601..3029
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2688..2707)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2898..2919
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3038..3058)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3043..3372
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3223..3358
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3285..3304
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3439..4233
                     /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(3493..3512)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4103..4122
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4407..4528
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4444..4463)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4498..4517
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4577..4593)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4577..4593)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4590..4612)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4601..4617
                     /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(4625..4631,4632..4649,4650..4655))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4670..4691
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4808..4825)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4979..5567)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5059..5078)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5738..6529,6530..6598))
                     /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     6361..6380
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6599..6703)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgcggcc
      901 gccaccatgg tgcggggttc tcatcatcat catcatcatg gtatggctag catgactggt
      961 ggacagcaaa tgggtcggga tctgtacgac gatgacgata aggatcccat gctgcgtcgc
     1021 gccgccctgg tggacggcgg caccggcggc agcgagctca gcgcgatcaa cgtgtatatc
     1081 atggctgaca aacagaagaa cggtatcaag gccaacttca agatccgcca caacatcgaa
     1141 gacggcggcg ttcagctggc tgatcactac cagcagaaca ccccaatcgg cgacggccct
     1201 gttctgctgc ctgacaacca ctacttaagc atccagtcta aactgtccaa agacccaaac
     1261 gagaagcgcg accacatggt tctgctggaa tttgttaccg ccgctggtat cacccacggc
     1321 atggacgaac tgtacaaggg cggcaccggg ggatctatgg tttccaaagg cgaggaactg
     1381 ttcactggtg ttgttcctat cctggttgaa ctggacggcg acgttaacgg ccacaaattc
     1441 agcgttagcg gggaaggcga aggagacgct acctacggca agctgactct gaagttcatc
     1501 tgcaccactg gcaagctgcc tgttccttgg cctaccctgg ttactacctt ctcctatggt
     1561 gttatggttt tcgccagata tcctgaccac atgaagcagc acgacttctt caagagcgcc
     1621 atgcctgagg gctacgttca ggaacgcacc atctttttca aggacgacgg caactataag
     1681 acacgcgctg aggttaagtt cgagggcgac actctggtta accgcatcga gctgaagggc
     1741 atcgacttca aggaggacgg caacatcctg ggccataagc ttgaatataa cagcatgcat
     1801 aagttttctc aagaacagat cggcgaaaac attgtgtgca gggtcatttg taccacgggt
     1861 caaattccca tccgagattt gtcagctgat atttcacaag tgcttaagga aaaacgatcc
     1921 ataaagaaag tttggacatt tggtagaaac ccagcctgtg actatcattt aggaaacatt
     1981 tcaagactgt caaataagca tttccaaata ctactaggag aagacggtaa ccttttattg
     2041 aatgacattt ccactaatgg gacctggtta aatgggcaaa aagtcgagaa gaacagcaat
     2101 cagttactgt ctcaaggtga tgaaataacc gttggtgtag gcgtggaatc agatatttta
     2161 tctctggtca ttttcataaa cgacaaattt aagcagtgcc tcgagcagaa caaagttgat
     2221 cgctaagaat tctgcagata tccatcacac tggcggccgc tcgagcatgc atctagaggg
     2281 ccctattcta tagtgtcacc taaatgctag agctcgctga tcagcctcga ctgtgccttc
     2341 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     2401 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     2461 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     2521 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     2581 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     2641 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     2701 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     2761 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     2821 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     2881 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     2941 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     3001 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt
     3061 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3121 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3181 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     3241 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     3301 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     3361 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca
     3421 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     3481 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     3541 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     3601 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc
     3661 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     3721 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     3781 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     3841 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     3901 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     3961 aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     4021 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     4081 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     4141 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     4201 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg
     4261 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa
     4321 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     4381 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     4441 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     4501 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     4561 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     4621 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     4681 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4741 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     4801 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     4861 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     4921 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     4981 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     5041 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     5101 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     5161 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     5221 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     5281 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     5341 acaggattag caaagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     5401 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     5461 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     5521 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     5581 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     5641 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     5701 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     5761 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     5821 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     5881 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     5941 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     6001 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     6061 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     6121 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     6181 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     6241 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     6301 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     6361 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     6421 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     6481 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     6541 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     6601 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     6661 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     6721 tgccacctga cgtc
//
