LOCUS       sequence-363362-        6749 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nuclear-targeted ExRai-AktAR2..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6749)
  AUTHORS   Chen M, Sun T, Zhong Y, Zhou X, Zhang J
  TITLE     A Highly Sensitive Fluorescent Akt Biosensor Reveals 
            Lysosome-Selective Regulation of Lipid Second Messengers and Kinase 
            Activity.
  JOURNAL   ACS Cent Sci. 2021 Dec 22;7(12):2009-2020. doi: 
            10.1021/acscentsci.1c00919. Epub 2021 Dec 3.
  PUBMED    34963894
REFERENCE   2  (bases 1 to 6749)
  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: 
            "10.1021/acscentsci.1c00919"; journalName: "ACS Cent Sci"; date: 
            "2021-12-22- 22"; volume: "7"; issue: "12"; pages: "2009-2020"
COMMENT     Sequence Label: pcDNA3-Nuc-ExRai-AktAR2
FEATURES             Location/Qualifiers
     source          1..6749
                     /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"
     primer_bind     1299..1320
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1426..1447)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1687..1706)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2254..2274
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(2302..2320)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2303..2320)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2340..2357)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2346..2570
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2616..3044
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2703..2722)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2913..2934
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3053..3073)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3058..3387
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3238..3373
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3300..3319
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3454..4248
                     /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(3508..3527)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4118..4137
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4422..4543
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4459..4478)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4513..4532
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4592..4608)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4592..4608)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4605..4627)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4616..4632
                     /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(4640..4646,4647..4664,4665..4670))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4685..4706
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4823..4840)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4994..5582)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5074..5093)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5753..6544,6545..6613))
                     /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     6376..6395
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6614..6718)
                     /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 aggatcccgt cgaccctcgt
     1021 ccgcgctcgt gcacctggcc ggaccccagg ccggagtttg gaggtaccgg cggcagcgag
     1081 ctcagcttgc agaacgtcta tatcaaggcc gacaagcaga agaacggcat caaggcgaac
     1141 ttcaagatcc gccacaacat cgaggacggc ggcgtgcagc tcgcctacca ctaccagcag
     1201 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcgtgcag
     1261 tccaaacttt cgaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     1321 accgccgccg ggatcactct cggcatggac gagctgtaca agggcggtac cggagggagc
     1381 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     1441 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gtgagggcga tgccacctac
     1501 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     1561 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     1621 cagcacgact tcttcaagtc cgccatgccc gaaggctaca tccaggagcg caccatcttc
     1681 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     1741 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     1801 aagctggagt acaacttgct gcataagttt tctcaagaac agatcggcga aaacattgtg
     1861 tgcagggtca tttgtaccac gggtcaaatt cccatccgag atttgtcagc tgatatttca
     1921 caagtgctta aggaaaaacg atccataaag aaagtttgga catttggtag aaacccagcc
     1981 tgtgactatc atttaggaaa catttcaaga ctgtcaaata agcatttcca aatactacta
     2041 ggagaagacg gtaacctttt attgaatgac atttccacta atgggacctg gttaaatggg
     2101 caaaaagtcg agaagaacag caatcagtta ctgtctcaag gtgatgaaat aaccgttggt
     2161 gtaggcgtgg aatcagatat tttatctctg gtcattttca taaacgacaa atttaagcag
     2221 tgcctcgagc agaacaaagt tgatcgcgaa ttccccaaaa agaagagaaa ggtggaagac
     2281 gcataatcta gagggcccta ttctatagtg tcacctaaat gctagagctc gctgatcagc
     2341 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     2401 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     2461 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     2521 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     2581 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     2641 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     2701 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     2761 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2821 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     2881 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     2941 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     3001 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
     3061 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3121 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     3181 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     3241 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     3301 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
     3361 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
     3421 tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     3481 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     3541 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     3601 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg
     3661 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     3721 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     3781 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     3841 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     3901 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     3961 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc
     4021 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     4081 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     4141 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     4201 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     4261 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     4321 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     4381 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     4441 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     4501 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     4561 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     4621 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     4681 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4741 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4801 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     4861 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4921 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4981 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     5041 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     5101 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     5161 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     5221 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     5281 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     5341 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     5401 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     5461 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5521 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5581 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5641 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     5701 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5761 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5821 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5881 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5941 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     6001 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     6061 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     6121 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     6181 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     6241 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     6301 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     6361 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     6421 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     6481 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     6541 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6601 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     6661 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6721 catttccccg aaaagtgcca cctgacgtc
//
