LOCUS       sequence-176157-        6254 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes C-terminal (substrate) fragment of bimolecular 
            super-resolution PKA activity reporter (bimFLINC-AKAR); targeted to 
            plasma membrane; use in conjunction with Dronpa-FHA1 (Addgene 
            #87711)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6254)
  AUTHORS   Mo GC, Ross B, Hertel F, Manna P, Yang X, Greenwald E, Booth C, 
            Plummer AM, Tenner B, Chen Z, Wang Y, Kennedy EJ, Cole PA, Fleming 
            KG, Palmer A, Jimenez R, Xiao J, Dedecker P, Zhang J
  TITLE     Genetically encoded biosensors for visualizing live-cell biochemical
            activity at super-resolution.
  JOURNAL   Nat Methods. 2017 Apr;14(4):427-434. doi: 10.1038/nmeth.4221. Epub 
            2017 Mar 13.
  PUBMED    28288122
REFERENCE   2  (bases 1 to 6254)
  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/nmeth.4221"; journalName: "Nat Methods"; date: "2017-04"; 
            volume: "14"; issue: "4"; pages: "427-434"
COMMENT     Sequence Label: PKAsub-TagRFPT-CAAX
FEATURES             Location/Qualifiers
     source          1..6254
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..18
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             22..54
                     /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(58..66,67..81)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             130..819
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTDMALK
                     LVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVARYC
                     DLPSKLGH"
     promoter        complement(886..904)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(887..904)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(924..941)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    930..1154
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1200..1628
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1287..1306)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1497..1518
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1637..1657)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1642..1971
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1822..1957
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1884..1903
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2038..2832
                     /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(2092..2111)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2702..2721
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3006..3127
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3043..3062)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3097..3116
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3176..3192)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3176..3192)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3189..3211)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3200..3216
                     /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(3224..3230,3231..3248,3249..3254))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3269..3290
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3407..3424)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3578..4166)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3658..3677)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4337..5128,5129..5197))
                     /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     4960..4979
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5198..5302)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5377..5396)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5568..5947
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5948..6151
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6102..6122
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     6196..6215
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6196..6214
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      6234..6243
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 catcatcatc atcatcatgg tatggctagc atgactggtg gacagcaaat gggtcgggat
       61 ctgtacgacg atgacgataa ggatcccgga ttgaggcgcg cgacgctggt tgacggcggc
      121 agcgagctca tgagcgagct gattaaggag aacatgcaca tgaagctgta catggagggc
      181 accgtgaaca accaccactt caagtgcaca tccgagggcg aaggcaagcc ctacgagggc
      241 acccagacca tgagaatcaa ggtggtcgag ggcggccctc tccccttcgc cttcgacatc
      301 ctggctacca gcttcatgta cggcagcaga accttcatca accacaccca gggcatcccc
      361 gatttcttta agcagtcctt ccctgagggc ttcacatggg agagagtcac cacatacgaa
      421 gacgggggcg tgctgaccgc tacccaggac accagcctcc aggacggctg cctcatctac
      481 aacgtcaaga tcagaggggt gaacttccca tccaacggcc ctgtgatgca gaagaaaaca
      541 ctcggctggg aggccaacac cgagatgctg taccccgctg acggcggcct ggaaggcaga
      601 accgacatgg ccctgaagct cgtgggcggg ggccacctga tctgcaactt caagaccaca
      661 tacagatcca agaaacccgc taagaacctc aagatgcccg gcgtctacta tgtggaccac
      721 agactggaaa gaatcaagga ggccgacaaa gagacctacg tcgagcagca cgaggtggct
      781 gtggccagat actgcgacct ccctagcaaa ctggggcaca aggaattcaa gaagaagaag
      841 aagagcaaga ccaagtgcgt gatcatgtaa tctagagggc cctattctat agtgtcacct
      901 aaatgctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
      961 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     1021 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     1081 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     1141 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     1201 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     1261 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     1321 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     1381 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     1441 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     1501 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     1561 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     1621 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     1681 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     1741 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     1801 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     1861 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag
     1921 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg
     1981 ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg
     2041 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     2101 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     2161 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag
     2221 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     2281 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     2341 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     2401 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     2461 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     2521 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc
     2581 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     2641 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     2701 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     2761 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     2821 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc
     2881 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     2941 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     3001 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     3061 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     3121 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     3181 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     3241 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     3301 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     3361 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     3421 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     3481 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     3541 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     3601 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     3661 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     3721 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     3781 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     3841 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     3901 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     3961 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     4021 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     4081 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     4141 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     4201 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     4261 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     4321 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     4381 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     4441 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     4501 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     4561 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     4621 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     4681 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     4741 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     4801 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     4861 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     4921 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     4981 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     5041 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     5101 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     5161 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     5221 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     5281 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat
     5341 cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata
     5401 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa
     5461 aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt
     5521 taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac attgattatt
     5581 gactagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
     5641 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
     5701 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
     5761 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
     5821 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
     5881 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
     5941 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
     6001 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
     6061 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
     6121 tgtacggtgg gaggtctata taagcagagc tctctggcta actagagaac ccactgctta
     6181 ctggcttatc gaaattaata cgactcacta tagggagacc caagcttgcg gccgccacca
     6241 tggtgcgggg ttct
//
