LOCUS       sequence-307498-        7782 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Full-length AKAP95 fused to FRB (rapamycin-binding domain). Used in 
            conjunction with pcDNA3-FKBP-ICUE3-NLS to monitor cAMP dynamics 
            within AKAP95 nanodomain..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7782)
  AUTHORS   Clister T, Greenwald EC, Baillie GS, Zhang J
  TITLE     AKAP95 Organizes a Nuclear Microdomain to Control Local cAMP for 
            Regulating Nuclear PKA.
  JOURNAL   Cell Chem Biol. 2019 Jun 20;26(6):885-891.e4. doi: 
            10.1016/j.chembiol.2019.03.003. Epub 2019 Apr 11.
  PUBMED    30982750
REFERENCE   2  (bases 1 to 7782)
  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: 
            "10.1016/j.chembiol.2019.03.003"; journalName: "Cell Chem Biol"; 
            date: "2019-06-20- 20"; volume: "26"; issue: "6"; pages: 
            "885-891.e4"
COMMENT     Sequence Label: pcDNA3.1(+)-AKAP95-FRB
FEATURES             Location/Qualifiers
     source          1..7782
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        130..509
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        510..713
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     664..684
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     758..777
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        758..776
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      790..799
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2881..3159
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     CDS             3166..3192
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(3230..3248)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3231..3248)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3268..3285)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3274..3498
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3544..3972
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3631..3650)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3841..3862
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3981..4001)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3986..4315
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4166..4301
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4228..4247
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4382..5176
                     /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(4436..4455)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5046..5065
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5350..5471
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5387..5406)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5441..5460
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5520..5536)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5520..5536)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5533..5555)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5544..5560
                     /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(5568..5574,5575..5592,5593..5598))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5613..5634
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5751..5768)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5922..6510)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6002..6021)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6681..7472,7473..7541))
                     /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     7304..7323
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7542..7646)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7721..7740)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt
       61 gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat
      121 atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag
      181 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
      241 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
      301 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
      361 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
      421 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
      481 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
      541 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
      601 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
      661 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc
      721 taactagaga acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga
      781 cccaagcttg ccaccatgga ccagggctac ggaggctacg gggcgtggag tgctggacct
      841 gccaacaccc agggtgcata tggaactggt gtggccagct ggcaaggtta tgaaaactac
      901 aattactatg gcgcccagaa caccagtgtc accacaggcg caacctacag ctacggccca
      961 gcctcgtggg aggccgccaa ggccaatgat ggcggcctgg cggccggggc ccctgccatg
     1021 cacatggcct cttacggccc agagccatgc accgacaatt ccgactccct cattgccaag
     1081 atcaaccagc gtttggacat gatgtccaag gaaggaggca ggggcgggag cggcggcggt
     1141 ggggagggca tacaggaccg ggagagctcc ttccgcttcc agccgttcga gtcctatgac
     1201 tccaggccct gcctgccgga gcacaacccc taccgcccca gctacagcta cgactatgag
     1261 ttcgacctgg ggtccgaccg caatggcagc tttggggggc agtacagtga atgccgagac
     1321 ccagcccggg agcggggctc ccttgatggc ttcatgcggg gccggggcca gggccgcttc
     1381 caggaccgga gcaaccctgg caccttcatg cgcagcgacc ccttcgtgcc ccccgctgcg
     1441 tcctctgagc ccctgtccac gccctggaac gagctgaact acgtgggtgg acggggcctg
     1501 ggagggccct cccccagccg gccacctccg tccctcttct cccagtccat ggctcccgac
     1561 tacggcgtga tgggcatgca gggggcgggc ggctatgaca gcaccatgcc ctacggatgt
     1621 ggccgctcgc agcctcggat gcgggatcgg gatcggccca agaggagagg gtttgaccgc
     1681 ttcggaccag atggcacggg caggaaacgg aagcagttcc aactttacga ggagccagac
     1741 accaaactgg cccgggttga cagtgaagga gatttctccg aaaatgatga cgcagctggt
     1801 gacttccgct caggagatga agaattcaag ggtgaggatg aactctgcga ctctgggagg
     1861 caaagaggag agaaggagga cgaggacgag gatgtgaaga agagaaggga aaagcaaagg
     1921 agaagagaca ggacgcggga ccgtgcagcc gacagaattc agtttgcctg ttctgtatgc
     1981 aagttccgta gctttgatga cgaagagatc cagaagcatc tgcaaagcaa atttcacaaa
     2041 gagaccctgc ggttcataag caccaagctg cccgacaaga ccgtggagtt cctccaggaa
     2101 tacattgtaa acagaaataa gaaaattgag aagcggcgtc aggaattgat ggagaaagaa
     2161 accgcaaaac caaaaccaga tcctttcaaa gggattggcc aggagcactt cttcaagaag
     2221 atcgaggctg ctcactgcct ggcctgcgac atgctaattc ctgcacagcc gcagctcctc
     2281 cagcggcacc tgcactccgt ggaccacaat cacaaccgca ggttggctgc tgaacagttc
     2341 aagaaaacca gtctccatgt ggctaagagt gttttgaaca acagacatat agtgaagatg
     2401 ctggaaaaat acctcaaggg tgaggaccct ttcaccagtg aaactgttga tccagaaatg
     2461 gaaggagatg acaatttagg aggtgaggat aagaaagaga cacctgagga ggtggccgcg
     2521 gacgtcttag cagaggtgat tacagcagca gtgagggccg tagatgggga aggagcgccc
     2581 gctccagaga gcagcgggga gccggctgag gacgaaggcc ccacggacac agcggaggcc
     2641 ggtagtgatc ctcaagccga acagctgctg gaagagcagg tgccctgtgg aacggcacat
     2701 gagaagggcg tccccaaggc cagaagtgag gctgcagagg ctggaaatgg cgccgagaca
     2761 atggcagcag aggcagaaag tgcccaaacc agagttgctc ctgccccagc tgccgcggat
     2821 gctgaagtgg aacaaactga tgcagagtct aaagacgctg ttcccacaga ggatcgatcc
     2881 atcctctggc atgagatgtg gcatgaaggc ctggaagagg catctcgttt gtactttggg
     2941 gaaaggaacg tgaaaggcat gtttgaggtg ctggagccct tgcatgctat gatggaacgg
     3001 ggcccccaga ctctgaagga aacatccttt aatcaggcct atggtcgaga tttaatggag
     3061 gcccaagagt ggtgcaggaa gtacatgaaa tcagggaatg tcaaggacct cctccaagcc
     3121 tgggacctct attatcatgt gttccgacga atctcaaaga ctagttatcc gtacgacgta
     3181 ccagactacg cataagcggc cgctcgagca tgcatctaga gggccctatt ctatagtgtc
     3241 acctaaatgc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     3301 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     3361 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     3421 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     3481 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     3541 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     3601 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     3661 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     3721 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     3781 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     3841 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     3901 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     3961 ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     4021 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     4081 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     4141 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     4201 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
     4261 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     4321 cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg
     4381 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     4441 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     4501 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     4561 gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     4621 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     4681 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     4741 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     4801 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     4861 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
     4921 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     4981 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     5041 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     5101 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     5161 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     5221 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     5281 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     5341 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     5401 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     5461 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     5521 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     5581 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     5641 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     5701 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     5761 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     5821 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     5881 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     5941 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     6001 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     6061 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     6121 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     6181 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     6241 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     6301 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     6361 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     6421 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     6481 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     6541 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     6601 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     6661 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     6721 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     6781 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     6841 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     6901 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     6961 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     7021 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     7081 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     7141 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     7201 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     7261 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     7321 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     7381 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     7441 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     7501 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     7561 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     7621 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac
     7681 ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg
     7741 catagttaag ccagtatctg ctccctgctt gtgtgttgga gg
//
