LOCUS       sequence-344936-        6392 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cytosol-targeted Akt-STOPS..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6392)
  AUTHORS   Zhou X, Zhong Y, Molinar-Inglis O, Kunkel MT, Chen M, Sun T, Zhang 
            J, Shyy JY, Trejo J, Newton AC, Zhang J
  TITLE     Location-specific inhibition of Akt reveals regulation of mTORC1 
            activity in the nucleus.
  JOURNAL   Nat Commun. 2020 Nov 30;11(1):6088. doi: 10.1038/s41467-020-19937-w.
  PUBMED    33257668
REFERENCE   2  (bases 1 to 6392)
  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 
            Commun."; date: "2020-11-30"; volume: "11(1):6088. doi"; pages: " 
            10.1038/s41467-020-19937-w"
COMMENT     Sequence Label: pcDNA3-Akt-STOPS-NES
FEATURES             Location/Qualifiers
     source          1..6392
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(56..75)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        247..626
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        627..830
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     781..801
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     875..894
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        875..893
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      913..922
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             934..951
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             955..987
                     /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(991..999,1000..1014)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             1021..1728
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1166..1184)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1434..1453
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1912..1938
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     promoter        complement(1957..1975)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1958..1975)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1995..2012)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2001..2225
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2271..2699
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2358..2377)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2568..2589
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2708..2728)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2713..3042
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2893..3028
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2955..2974
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3109..3903
                     /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(3163..3182)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3773..3792
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4077..4198
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4114..4133)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4168..4187
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4247..4263)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4247..4263)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4260..4282)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4271..4287
                     /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(4295..4301,4302..4319,4320..4325))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4340..4361
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4478..4495)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4649..5237)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4729..4748)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5408..6199,6200..6268))
                     /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     6031..6050
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6269..6373)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
       61 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
      121 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
      181 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
      241 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
      301 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      361 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      421 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      481 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      541 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      601 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      661 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      721 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
      781 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa
      841 ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc
      901 aagcttgcgg ccgccaccat ggtgcggggt tctcatcatc atcatcatca tggtatggct
      961 agcatgactg gtggacagca aatgggtcgg gatctgtacg acgatgacga taaggatccc
     1021 atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag
     1081 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc
     1141 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
     1201 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac
     1261 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc
     1321 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac
     1381 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta
     1441 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc
     1501 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct
     1561 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc
     1621 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa
     1681 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagcg catgcatcct
     1741 cgtccgcgct cgtgcacctg gccggacccc aggccggagt ttggagtcga ccctcgtccg
     1801 cgctcgtgca cctggccgga ccccaggccg gagtttggag gtaccggcgg cagcgagctc
     1861 cctcgtccgc gctcgtgcac ctggccggac cccaggccgg agtttgaatt cctgcccccc
     1921 ctggagcgcc tgaccctgta atctagaggg ccctattcta tagtgtcacc taaatgctag
     1981 agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     2041 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     2101 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     2161 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     2221 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
     2281 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     2341 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     2401 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     2461 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     2521 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     2581 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     2641 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta
     2701 attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     2761 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     2821 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     2881 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     2941 tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag
     3001 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt
     3061 atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
     3121 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     3181 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     3241 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
     3301 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     3361 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     3421 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     3481 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     3541 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     3601 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc
     3661 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     3721 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     3781 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     3841 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     3901 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag
     3961 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     4021 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
     4081 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     4141 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     4201 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     4261 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     4321 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     4381 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     4441 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     4501 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     4561 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     4621 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     4681 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     4741 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4801 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4861 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4921 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4981 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     5041 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     5101 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     5161 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     5221 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     5281 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     5341 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     5401 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     5461 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     5521 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     5581 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     5641 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     5701 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     5761 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5821 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5881 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5941 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     6001 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     6061 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     6121 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     6181 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     6241 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     6301 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     6361 taggggttcc gcgcacattt ccccgaaaag tg
//
