LOCUS       sequence-230229-        6671 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  cp-T-sapphire-based single-color PKA biosensor targeted to the 
            plasma membrane..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6671)
  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 6671)
  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-Lyn-sapphireAKAR
FEATURES             Location/Qualifiers
     source          1..6671
                     /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"
     promoter        complement(2224..2242)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2225..2242)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2262..2279)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2268..2492
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2538..2966
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2625..2644)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2835..2856
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2975..2995)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2980..3309
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3160..3295
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3222..3241
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3376..4170
                     /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(3430..3449)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4040..4059
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4344..4465
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4381..4400)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4435..4454
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4514..4530)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4514..4530)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4527..4549)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4538..4554
                     /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(4562..4568,4569..4586,4587..4592))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4607..4628
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4745..4762)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4916..5504)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4996..5015)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5675..6466,6467..6535))
                     /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     6298..6317
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6536..6640)
                     /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 gctgcatcaa gagcaagcgc aaggacaagg atcccatgct gcgtcgcgcc
      961 accctggtgg acggcggcac cggcggcagc gagctcagcg cgatcaacgt gtatatcatg
     1021 gctgacaaac agaagaacgg tatcaaggcc aacttcaaga tccgccacaa catcgaagac
     1081 ggcggcgttc agctggctga tcactaccag cagaacaccc caatcggcga cggccctgtt
     1141 ctgctgcctg acaaccacta cttaagcatc cagtctaaac tgtccaaaga cccaaacgag
     1201 aagcgcgacc acatggttct gctggaattt gttaccgccg ctggtatcac ccacggcatg
     1261 gacgaactgt acaagggcgg caccggggga tctatggttt ccaaaggcga ggaactgttc
     1321 actggtgttg ttcctatcct ggttgaactg gacggcgacg ttaacggcca caaattcagc
     1381 gttagcgggg aaggcgaagg agacgctacc tacggcaagc tgactctgaa gttcatctgc
     1441 accactggca agctgcctgt tccttggcct accctggtta ctaccttctc ctatggtgtt
     1501 atggttttcg ccagatatcc tgaccacatg aagcagcacg acttcttcaa gagcgccatg
     1561 cctgagggct acgttcagga acgcaccatc tttttcaagg acgacggcaa ctataagaca
     1621 cgcgctgagg ttaagttcga gggcgacact ctggttaacc gcatcgagct gaagggcatc
     1681 gacttcaagg aggacggcaa catcctgggc cataagcttg aatataacag catgcataag
     1741 ttttctcaag aacagatcgg cgaaaacatt gtgtgcaggg tcatttgtac cacgggtcaa
     1801 attcccatcc gagatttgtc agctgatatt tcacaagtgc ttaaggaaaa acgatccata
     1861 aagaaagttt ggacatttgg tagaaaccca gcctgtgact atcatttagg aaacatttca
     1921 agactgtcaa ataagcattt ccaaatacta ctaggagaag acggtaacct tttattgaat
     1981 gacatttcca ctaatgggac ctggttaaat gggcaaaaag tcgagaagaa cagcaatcag
     2041 ttactgtctc aaggtgatga aataaccgtt ggtgtaggcg tggaatcaga tattttatct
     2101 ctggtcattt tcataaacga caaatttaag cagtgcctcg agcagaacaa agttgatcgc
     2161 taagaattct gcagatatcc atcacactgg cggccgctcg agcatgcatc tagagggccc
     2221 tattctatag tgtcacctaa atgctagagc tcgctgatca gcctcgactg tgccttctag
     2281 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     2341 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     2401 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     2461 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     2521 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     2581 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     2641 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     2701 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     2761 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     2821 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     2881 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     2941 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga
     3001 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     3061 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3121 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     3181 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     3241 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     3301 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca agagacagga
     3361 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     3421 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     3481 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     3541 gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     3601 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     3661 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     3721 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     3781 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     3841 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     3901 gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     3961 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     4021 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     4081 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     4141 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     4201 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     4261 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     4321 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     4381 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     4441 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     4501 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     4561 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     4621 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4681 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     4741 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     4801 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     4861 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     4921 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     4981 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5041 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5101 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5161 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5221 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     5281 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5341 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5401 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     5461 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5521 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5581 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     5641 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     5701 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     5761 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     5821 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     5881 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     5941 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     6001 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     6061 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6121 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6181 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6241 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6301 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6361 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6421 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6481 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6541 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6601 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     6661 cacctgacgt c
//
