LOCUS       sequence-344969-        6395 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nuclear-targeted Akt-STOPS..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6395)
  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 6395)
  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-NLS
FEATURES             Location/Qualifiers
     source          1..6395
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(20..39)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        211..590
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        591..794
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     745..765
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     839..858
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        839..857
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      877..886
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             898..915
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             919..951
                     /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(955..963,964..978)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             985..1692
                     /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(1130..1148)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1398..1417
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1876..1896
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(1924..1942)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1925..1942)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1962..1979)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1968..2192
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2238..2666
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2325..2344)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2535..2556
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2675..2695)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2680..3009
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2860..2995
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2922..2941
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3076..3870
                     /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(3130..3149)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3740..3759
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4044..4165
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4081..4100)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4135..4154
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4214..4230)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4214..4230)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4227..4249)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4238..4254
                     /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(4262..4268,4269..4286,4287..4292))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4307..4328
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4445..4462)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4616..5204)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4696..4715)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5375..6166,6167..6235))
                     /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     5998..6017
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6236..6340)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc
       61 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa
      121 ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc
      181 ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt
      241 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
      301 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
      361 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
      421 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
      481 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
      541 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
      601 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
      661 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
      721 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
      781 atataagcag agctctctgg ctaactagag aacccactgc ttactggctt atcgaaatta
      841 atacgactca ctatagggag acccaagctt gcggccgcca ccatggtgcg gggttctcat
      901 catcatcatc atcatggtat ggctagcatg actggtggac agcaaatggg tcgggatctg
      961 tacgacgatg acgataagga tcccatggtg agcaagggcg aggaggataa catggccatc
     1021 atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc
     1081 gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag
     1141 gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac
     1201 ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc
     1261 cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg
     1321 acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc
     1381 aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc
     1441 gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg
     1501 aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg
     1561 cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac
     1621 tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac
     1681 gagctgtaca agcgcatgca tcctcgtccg cgctcgtgca cctggccgga ccccaggccg
     1741 gagtttggag tcgaccctcg tccgcgctcg tgcacctggc cggaccccag gccggagttt
     1801 ggaggtaccg gcggcagcga gctccctcgt ccgcgctcgt gcacctggcc ggaccccagg
     1861 ccggagtttg aattccccaa aaagaagaga aaggtggaag acgcataatc tagagggccc
     1921 tattctatag tgtcacctaa atgctagagc tcgctgatca gcctcgactg tgccttctag
     1981 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     2041 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     2101 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     2161 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     2221 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     2281 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     2341 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     2401 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     2461 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     2521 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     2581 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     2641 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga
     2701 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2761 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     2821 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     2881 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     2941 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     3001 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca agagacagga
     3061 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     3121 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     3181 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     3241 gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     3301 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     3361 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     3421 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     3481 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     3541 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     3601 gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     3661 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     3721 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     3781 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     3841 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     3901 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     3961 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     4021 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     4081 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     4141 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     4201 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     4261 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     4321 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4381 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     4441 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     4501 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     4561 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     4621 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     4681 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     4741 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     4801 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     4861 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     4921 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     4981 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5041 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5101 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     5161 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5221 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5281 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     5341 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     5401 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     5461 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     5521 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     5581 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     5641 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     5701 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     5761 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     5821 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     5881 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     5941 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6001 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6061 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6121 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6181 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6241 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6301 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     6361 cacctgacgt cgacggatcg ggagatctcc cgatc
//
