LOCUS       sequence-211071-        7651 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expression of human eEF2K (S441A/S445A) in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7651)
  AUTHORS   Kruiswijk F, Yuniati L, Magliozzi R, Low TY, Lim R, Bolder R, 
            Mohammed S, Proud CG, Heck AJ, Pagano M, Guardavaccaro D
  TITLE     Coupled activation and degradation of eEF2K regulates protein 
            synthesis in response to genotoxic stress.
  JOURNAL   Sci Signal. 2012 Jun 5;5(227):ra40. doi: 10.1126/scisignal.2002718.
  PUBMED    22669845
REFERENCE   2  (bases 1 to 7651)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci 
            Signal."; date: "2012-06-5"; volume: "5(227):ra40. doi"; pages: " 
            10.1126/scisignal.2002718"
COMMENT     Sequence Label: pcDNA3-HA-eEF2K(S441A/S445A)
FEATURES             Location/Qualifiers
     source          1..7651
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(40..59)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        231..610
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        611..814
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     765..785
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     859..878
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        859..877
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             922..948
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(3238..3255)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3244..3468
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3514..3942
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3601..3620)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3811..3832
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3951..3971)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3956..4285
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4136..4271
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4198..4217
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4352..5146
                     /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(4406..4425)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5016..5035
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5320..5441
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5357..5376)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5411..5430
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5490..5506)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5490..5506)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5503..5525)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5514..5530
                     /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(5538..5544,5545..5562,5563..5568))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5583..5604
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5721..5738)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5892..6480)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5972..5991)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6651..7442,7443..7511))
                     /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     7274..7293
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7512..7616)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc
       61 atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag
      121 caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag
      181 ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt
      241 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
      301 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
      361 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
      421 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
      481 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
      541 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
      601 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
      661 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
      721 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
      781 gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc
      841 ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg gctagcgttt
      901 aaacttaagc ttaccatggg atacccatac gatgttccag attacgctgt ggtacctagg
      961 atccagtgtg gtggaattcg gcttatggca gacgaagacc tcatcttccg cctggaaggc
     1021 gttgatggcg gccagtcccc ccgagctggc catgatggtg attctgatgg ggacagcgac
     1081 gatgaggaag gttacttcat ctgccccatc acggatgacc caagctcgaa ccagaatgtc
     1141 aattccaagg ttaataagta ctacagcaac ctaacaaaaa gtgagcggta tagctccagc
     1201 gggtccccgg caaactcctt ccacttcaag gaagcctgga agcacgcaat ccagaaggcc
     1261 aagcacatgc ccgacccctg ggctgagttc cacctggaag atattgccac cgaacgtgct
     1321 actcgacaca ggtacaacgc cgtcaccggg gaatggctgg atgatgaagt tctgatcaag
     1381 atggcatctc agcccttcgg ccgaggagca atgagggagt gcttccggac gaagaagctc
     1441 tccaacttct tgcatgccca gcagtggaag ggagcctcca actacgtggc gaagcgctac
     1501 atcgagcccg tagaccggga tgtgtacttt gaggacgtgc gtctacagat ggaggccaag
     1561 ctctgggggg aggagtataa tcggcacaag ccccccaagc aggtggacat catgcagatg
     1621 tgcatcatcg agctgaagga cagaccgggc aagcccctct tccacctgga gcactacatc
     1681 gagggcaagt acatcaagta caactccaac tctggctttg tccgcgatga caacatccgc
     1741 ctgacgccgc aggccttcag ccacttcact tttgagcgtt ccggccatca gctgatagtg
     1801 gtggacatcc agggagttgg ggatctctac actgacccac agatccacac ggagacgggc
     1861 actgactttg gagacggcaa cctaggtgtc cgcgggatgg cgctcttctt ctactctcat
     1921 gcctgcaacc ggatttgcga gagcatgggc cttgctccct ttgacctctc gccccgggag
     1981 agggatgcag tgaatcagaa caccaagctg ctgcaatcag ccaagaccat cttgagagga
     2041 acagaggaaa aatgtgggag cccccgagta aggaccctct ctgggagccg gccacccctg
     2101 ctccgtcccc tttcagagaa ctctggagac gagaacatga gcgacgtgac cttcgactct
     2161 ctcccttctt ccccatcttc ggccacacca cacagccaga agctagacca cctccattgg
     2221 ccagtgttca gtgacctcga taacatggca tccagagacc atgatcatct agacaaccac
     2281 cgggagtctg agaatagtgg ggacgccgga taccccgctg agaagcgggg tgagctggat
     2341 gaccctgagc cccgagaaca tggccactca tacagtaatc ggaagtacga gtctgacgaa
     2401 gacagcctgg gcagctctgg acgggtatgt gtagagaagt ggaatctcct caactcctcc
     2461 cgcctccacc tgccgagggc ttcggccgtg gccctggaag tgcaaaggct taatgctctg
     2521 gacctcgaaa agaaaatcgg gaagtccatt ttggggaagg tccatctggc catggtgcgc
     2581 taccacgagg gtgggcgctt ctgcgagaag ggcgaggagt gggaccagga gtcggctgtc
     2641 ttccacctgg agcacgcagc caacctgggc gagctggagg ccatcgtggg cctggggctc
     2701 atgtactcgc agttgcctca tcacatccta gccgatgtct ctctgaagga gacagaagag
     2761 aacaaaacca aaggatttga ttacttacta aaggccgctg aagctggcga caggcagtcc
     2821 atgatcctag tggcgcgagc ttttgactct ggccagaacc tcagcccgga caggtgccaa
     2881 gactggctag aggccctgca ctggtacaac actgccctgg agatgacgga ctgtgatgag
     2941 ggcggtgagt acgacggaat gcaggacgag ccccggtaca tgatgctggc cagggaggcc
     3001 gagatgctgt tcacaggagg ctacgggctg gagaaggacc cgcagagatc aggggacttg
     3061 tatacccagg cagcagaggc agcgatggaa gccatgaagg gccgactggc caaccagtac
     3121 taccaaaagg ctgaagaggc ctgggcccaa atggaggaat aaaagccgaa ttctgcagat
     3181 atccagcaca gtggcggccg ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct
     3241 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     3301 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     3361 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     3421 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
     3481 aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg
     3541 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     3601 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     3661 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     3721 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     3781 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     3841 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     3901 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg
     3961 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     4021 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     4081 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     4141 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     4201 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt
     4261 ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc
     4321 tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     4381 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     4441 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     4501 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct
     4561 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     4621 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     4681 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     4741 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     4801 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     4861 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     4921 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     4981 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     5041 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     5101 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     5161 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     5221 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     5281 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     5341 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     5401 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     5461 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     5521 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     5581 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     5641 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5701 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5761 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5821 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5881 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5941 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     6001 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     6061 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     6121 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     6181 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     6241 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     6301 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     6361 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     6421 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     6481 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     6541 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     6601 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     6661 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     6721 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     6781 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     6841 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     6901 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     6961 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     7021 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     7081 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     7141 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     7201 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     7261 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     7321 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     7381 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     7441 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     7501 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     7561 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     7621 tttccccgaa aagtgccacc tgacgtcgac g
//
