LOCUS       sequence-343410-        6404 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Negative-control construct for Akt-STOPS..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6404)
  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 6404)
  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-Scramble
FEATURES             Location/Qualifiers
     source          1..6404
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(39..58)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        230..609
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        610..813
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     764..784
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     858..877
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        858..876
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      896..905
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             917..934
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             938..970
                     /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(974..982,983..997)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             1004..1711
                     /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(1149..1167)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1417..1436
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        complement(1952..1970)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1953..1970)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1990..2007)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1996..2220
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2266..2694
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2353..2372)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2563..2584
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2703..2723)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2708..3037
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2888..3023
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2950..2969
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3104..3898
                     /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(3158..3177)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3768..3787
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4072..4193
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4109..4128)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4163..4182
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4242..4258)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4242..4258)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4255..4277)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4266..4282
                     /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(4290..4296,4297..4314,4315..4320))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4335..4356
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4473..4490)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4644..5232)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4724..4743)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5403..6194,6195..6263))
                     /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     6026..6045
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6264..6368)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca
       61 tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc
      121 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg
      181 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
      241 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      301 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      361 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      421 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      481 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      541 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      601 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      661 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      721 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      781 cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga acccactgct
      841 tactggctta tcgaaattaa tacgactcac tatagggaga cccaagcttg cggccgccac
      901 catggtgcgg ggttctcatc atcatcatca tcatggtatg gctagcatga ctggtggaca
      961 gcaaatgggt cgggatctgt acgacgatga cgataaggat cccatggtga gcaagggcga
     1021 ggaggataac atggccatca tcaaggagtt catgcgcttc aaggtgcaca tggagggctc
     1081 cgtgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac
     1141 ccagaccgcc aagctgaagg tgaccaaggg tggccccctg cccttcgcct gggacatcct
     1201 gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag caccccgccg acatccccga
     1261 ctacttgaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga
     1321 cggcggcgtg gtgaccgtga cccaggactc ctccctgcag gacggcgagt tcatctacaa
     1381 ggtgaagctg cgcggcacca acttcccctc cgacggcccc gtaatgcaga agaagaccat
     1441 gggctgggag gcctcctccg agcggatgta ccccgaggac ggcgccctga agggcgagat
     1501 caagcagagg ctgaagctga aggacggcgg ccactacgac gctgaggtca agaccaccta
     1561 caaggccaag aagcccgtgc agctgcccgg cgcctacaac gtcaacatca agttggacat
     1621 cacctcccac aacgaggact acaccatcgt ggaacagtac gaacgcgccg agggccgcca
     1681 ctccaccggc ggcatggacg agctgtacaa gcgcatgcat accccctgcc cgagcgagtg
     1741 gccgcgccgc ccgcgcgatc catttggagt cgacaccccc tgcccgagcg agtggccgcg
     1801 ccgcccgcgc gatccatttg gaggtaccgg cggcagcgag ctcaccccct gcccgagcga
     1861 gtggccgcgc cgcccgcgcg atccatttta agaattctgc agatatccat cacactggcg
     1921 gccgctcgag catgcatcta gagggcccta ttctatagtg tcacctaaat gctagagctc
     1981 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     2041 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     2101 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     2161 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     2221 cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg
     2281 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     2341 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     2401 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     2461 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     2521 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     2581 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     2641 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct
     2701 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     2761 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     2821 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     2881 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     2941 aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta
     3001 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc
     3061 cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
     3121 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     3181 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     3241 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg
     3301 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     3361 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     3421 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     3481 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     3541 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     3601 gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt
     3661 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     3721 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     3781 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     3841 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc
     3901 gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc
     3961 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     4021 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt
     4081 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     4141 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     4201 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
     4261 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     4321 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     4381 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4441 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     4501 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4561 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4621 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4681 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4741 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     4801 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     4861 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     4921 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     4981 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     5041 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     5101 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     5161 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     5221 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5281 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     5341 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     5401 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     5461 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     5521 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     5581 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     5641 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     5701 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     5761 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     5821 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     5881 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     5941 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     6001 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     6061 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     6121 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     6181 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     6241 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     6301 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     6361 gttccgcgca catttccccg aaaagtgcca cctgacgtcg acgg
//
