LOCUS       sequence-307044-        7490 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cyan-fluorescent homoFRET/anisotropy-based biosensor for monitoring 
            Protein Kinase A activity. Contains monomeric 
            Cerulean3/cpCerulean3-E173 homoFRET pair..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7490)
  AUTHORS   Ross BL, Tenner B, Markwardt ML, Zviman A, Shi G, Kerr JP, Snell NE,
            McFarland JJ, Mauban JR, Ward CW, Rizzo MA, Zhang J
  TITLE     Single-color, ratiometric biosensors for detecting signaling 
            activities in live cells.
  JOURNAL   eLife. 2018 Jul 3;7. pii: 35458. doi: 10.7554/eLife.35458
  PUBMED    29968564
REFERENCE   2  (bases 1 to 7490)
  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: "eLife."; 
            date: "2018-07-3"; volume: "7. pii: 35458. doi"; pages: " 
            10.7554/eLife.3545"
COMMENT     Sequence Label: pcDNA3-Cerulean3-cpCerulean3-FLARE-AKAR
FEATURES             Location/Qualifiers
     source          1..7490
                     /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"
     CDS             join(1009..1011,1012..1014,1015..1725)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAIHGNVYITADKQKNGIK
                     ANFGLNCNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     promoter        complement(3043..3061)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3044..3061)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3081..3098)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3087..3311
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3357..3785
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3444..3463)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3654..3675
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3794..3814)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3799..4128
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3979..4114
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4041..4060
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4195..4989
                     /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(4249..4268)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4859..4878
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5163..5284
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5200..5219)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5254..5273
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5333..5349)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5333..5349)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5346..5368)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5357..5373
                     /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(5381..5387,5388..5405,5406..5411))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5426..5447
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5564..5581)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5735..6323)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5815..5834)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6494..7285,7286..7354))
                     /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     7117..7136
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7355..7459)
                     /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 ggtgagcaag
     1021 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     1081 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     1141 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     1201 ctgagctggg gcgtgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc
     1261 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     1321 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     1381 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     1441 aacgccatcc acggcaacgt ctatatcacc gccgacaagc agaagaacgg catcaaggcc
     1501 aacttcggcc tcaactgcaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     1561 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     1621 cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     1681 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagcgcat gcataagttt
     1741 tctcaagaac agatcggcga aaacattgtg tgcagggtca tttgtaccac gggtcaaatt
     1801 cccatccgag atttgtcagc tgatatttca caagtgctta aggaaaaacg atccataaag
     1861 aaagtttgga catttggtag aaacccagcc tgtgactatc atttaggaaa catttcaaga
     1921 ctgtcaaata agcatttcca aatactacta ggagaagacg gtaacctttt attgaatgac
     1981 atttccacta atgggacctg gttaaatggg caaaaagtcg agaagaacag caatcagtta
     2041 ctgtctcaag gtgatgaaat aaccgttggt gtaggcgtgg aatcagatat tttatctctg
     2101 gtcattttca taaacgacaa atttaagcag tgcctcgagc agaacaaagt tgatcgctct
     2161 gcaggtaagc caggcagcgg cgagggcagc accaagggcc tgcgtcgcgc caccctggtg
     2221 gacggcggca ccggcggcag cgagctcgac ggcagcgtgc agctcgccga ccactaccag
     2281 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     2341 cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     2401 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagggtgg cagcggtggc
     2461 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     2521 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     2581 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     2641 ctcgtgacca ccctgagctg gggcgtgcag tgcttcgccc gctaccccga ccacatgaag
     2701 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     2761 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     2821 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     2881 aagctggagt acaacgccat ccacggcaac gtctatatca ccgccgacaa gcagaagaac
     2941 ggcatcaagg ccaacttcgg cctcaactgc aacatcgagt aagaattctg cagatatcca
     3001 tcacactggc ggccgctcga gcatgcatct agagggccct attctatagt gtcacctaaa
     3061 tgctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     3121 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     3181 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     3241 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     3301 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     3361 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     3421 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     3481 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     3541 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     3601 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     3661 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     3721 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     3781 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     3841 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     3901 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     3961 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     4021 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta
     4081 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga
     4141 gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt
     4201 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     4261 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     4321 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
     4381 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     4441 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     4501 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     4561 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     4621 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     4681 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
     4741 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     4801 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     4861 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     4921 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     4981 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     5041 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     5101 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     5161 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     5221 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     5281 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     5341 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     5401 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     5461 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5521 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     5581 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     5641 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     5701 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     5761 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     5821 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5881 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     5941 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     6001 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     6061 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     6121 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     6181 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     6241 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     6301 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     6361 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     6421 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     6481 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     6541 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     6601 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     6661 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     6721 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     6781 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     6841 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     6901 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     6961 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     7021 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     7081 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     7141 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     7201 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     7261 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     7321 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     7381 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     7441 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
