LOCUS       sequence-363390-        6746 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cytosol-targeted ExRai-AktAR2..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6746)
  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 6746)
  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-Cyto-ExRai-AktAR2
FEATURES             Location/Qualifiers
     source          1..6746
                     /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..2280
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     promoter        complement(2299..2317)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2300..2317)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2337..2354)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2343..2567
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2613..3041
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2700..2719)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2910..2931
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3050..3070)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3055..3384
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3235..3370
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3297..3316
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3451..4245
                     /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(3505..3524)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4115..4134
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4419..4540
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4456..4475)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4510..4529
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4589..4605)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4589..4605)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4602..4624)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4613..4629
                     /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(4637..4643,4644..4661,4662..4667))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4682..4703
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4820..4837)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4991..5579)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5071..5090)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5750..6541,6542..6610))
                     /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     6373..6392
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6611..6715)
                     /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 ttcctgcccc ccctggagcg cctgaccctg
     2281 taatctagag ggccctattc tatagtgtca cctaaatgct agagctcgct gatcagcctc
     2341 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     2401 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     2461 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     2521 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     2581 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     2641 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     2701 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     2761 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     2821 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     2881 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     2941 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     3001 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     3061 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3121 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3181 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     3241 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     3301 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     3361 tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct
     3421 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     3481 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     3541 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     3601 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     3661 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     3721 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     3781 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     3841 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     3901 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     3961 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     4021 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     4081 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     4141 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     4201 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     4261 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     4321 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     4381 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     4441 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     4501 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     4561 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     4621 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     4681 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4741 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4801 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     4861 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     4921 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4981 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5041 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5101 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5161 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     5221 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     5281 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     5341 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5401 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     5461 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     5521 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     5581 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     5641 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     5701 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     5761 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     5821 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     5881 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     5941 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     6001 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6061 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     6121 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     6181 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     6241 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     6301 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6361 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6421 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     6481 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     6541 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     6601 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     6661 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     6721 ttccccgaaa agtgccacct gacgtc
//
