LOCUS       sequence-425970-        7179 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of full-length human EIF4A2. C-terminal NanoLuc
            luciferase tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7179)
  AUTHORS   Ackloo S, Li F, Szewczyk M, Seitova A, Loppnau P, Zeng H, Xu J, 
            Ahmad S, Arnautova YA, Baghaie AJ, Beldar S, Bolotokova A, Centrella
            PA, Chau I, Clark MA, Cuozzo JW, Dehghani-Tafti S, Disch JS, Dong A,
            Dumas A, Feng JA, Ghiabi P, Gibson E, Gilmer J, Goldman B, Green SR,
            Guie MA, Guilinger JP, Harms N, Herasymenko O, Houliston S, 
            Hutchinson A, Kearnes S, Keefe AD, Kimani SW, Kramer T, Kutera M, 
            Kwak HA, Lento C, Li Y, Liu J, Loup J, Machado RAC, Mulhern CJ, 
            Perveen S, Righetto GL, Riley P, Shrestha S, Sigel EA, Silva M, 
            Sintchak MD, Slakman BL, Taylor RD, Thompson J, Torng W, 
            Underkoffler C, von Rechenberg M, Walsh RT, Watson I, Wilson DJ, 
            Wolf E, Yadav M, Yazdi AK, Zhang J, Zhang Y, Santhakumar V, Edwards 
            AM, Barsyte-Lovejoy D, Schapira M, Brown PJ, Halabelian L, 
            Arrowsmith CH
  TITLE     A Target Class Ligandability Evaluation of WD40 Repeat-Containing 
            Proteins.
  JOURNAL   J Med Chem. 2025 Jan 23;68(2):1092-1112. doi: 
            10.1021/acs.jmedchem.4c02010. Epub 2024 Nov 4.
  PUBMED    39495097
REFERENCE   2  (bases 1 to 7179)
  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/acs.jmedchem.4c02010"; journalName: "J Med Chem"; date: 
            "2025-01-23- 23"; volume: "68"; issue: "2"; pages: "1092-1112"
COMMENT     Sequence Label: pNLF1-C_EIF4A2:1-407
FEATURES             Location/Qualifiers
     source          1..7179
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..721
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     672..692
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          857..989
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     972..991
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     1033..1052
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1033..1051
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2354..2866
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     primer_bind     complement(2942..2960)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    2997..3118
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3008..3027)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3062..3081
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3363..3720
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3363..3554
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3386..3406)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3571..3706
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3751..4788
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             4767..4775
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     polyA_signal    4812..4860
                     /note="synthetic polyadenylation signal"
     promoter        5016..5120
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5121..5189,5190..5981)
                     /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     complement(5339..5358)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(6139..6727)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6219..6238)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    6843..7126
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      661 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      721 tggtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc acagttaaat
      781 tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca gaagttggtc
      841 gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa
      901 actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac
      961 tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta
     1021 aggctagagt attaatacga ctcactatag ggctagcaaa gcgatcgctt ccgaattcgc
     1081 caccatgtct ggtggctccg cggattataa cagagaacat ggcggcccag agggaatgga
     1141 ccccgatggt gtcatcgaga gcaactggaa tgagattgtt gataactttg atgatatgaa
     1201 tttaaaggag tctctccttc gtggcatcta tgcttacggt tttgagaagc cttccgctat
     1261 tcagcagaga gctattattc cctgtattaa agggtatgat gtgattgctc aagctcagtc
     1321 aggtactggc aagacagcca catttgctat ttccatcctg caacagttgg agattgagtt
     1381 caaggagacc caagcactag tattggcccc caccagagaa ctggctcaac agatccaaaa
     1441 ggtaattctg gcacttggag actatatggg agccacttgt catgcctgca ttggtggaac
     1501 aaatgttcga aatgaaatgc aaaaactgca ggctgaagca ccacatattg ttgttggtac
     1561 acccgggaga gtgtttgata tgttaaacag aagatacctt tctccaaaat ggatcaaaat
     1621 gtttgttttg gatgaagcag atgaaatgtt gagccgtggt tttaaggatc aaatctatga
     1681 gattttccaa aaactaaaca caagtattca ggttgtgttg ctttctgcca caatgccaac
     1741 tgatgtgttg gaagtgacca aaaaattcat gagagatcca attcgaattc tggtgaaaaa
     1801 ggaagaattg acccttgaag gaatcaaaca gttttatatt aatgttgaga gagaggaatg
     1861 gaagttggat acactttgtg acttgtacga gacactgacc attacacagg ctgttatttt
     1921 tctcaatacg aggcgcaagg tggactggct gactgagaag atgcatgcca gagacttcac
     1981 agtttctgct ctgcatggtg acatggacca gaaggagaga gatgttatca tgagggaatt
     2041 ccggtcaggg tcaagtcgtg ttctgatcac tactgacttg ttggctcgcg ggattgatgt
     2101 gcaacaagtg tctttggtta taaattatga tctacctacc aatcgtgaaa actatattca
     2161 cagaattggc agagggggtc gatttgggag gaaaggtgtg gctataaact ttgttactga
     2221 agaagacaag aggattcttc gtgacattga gactttctac aatactacag tggaggagat
     2281 gcccatgaat gtggctgacc ttatttctag atttgggccc aattcctgca ggcgagctct
     2341 cggctcgagc ggcgtcttca cactcgaaga tttcgttggg gactggcgac agacagccgg
     2401 ctacaacctg gaccaagtcc ttgaacaggg aggtgtgtcc agtttgtttc agaatctcgg
     2461 ggtgtccgta actccgatcc aaaggattgt cctgagcggt gaaaatgggc tgaagatcga
     2521 catccatgtc atcatcccgt atgaaggtct gagcggcgac caaatgggcc agatcgaaaa
     2581 aatttttaag gtggtgtacc ctgtggatga tcatcacttt aaggtgatcc tgcactatgg
     2641 cacactggta atcgacgggg ttacgccgaa catgatcgac tatttcggac ggccgtatga
     2701 aggcatcgcc gtgttcgacg gcaaaaagat cactgtaaca gggaccctgt ggaacggcaa
     2761 caaaattatc gacgagcgcc tgatcaaccc cgacggctcc ctgctgttcc gagtaaccat
     2821 caacggagtg accggctggc ggctgtgcga acgcattctg gcgtaaggcc gcgactctag
     2881 ggcatgcaag ctgatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
     2941 accgctgagc aataactagc ataacccctt ggggcggccg cttcgagcag acatgataag
     3001 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     3061 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa
     3121 caacaacaat tgcattcatt ttatgtttca ggttcagggg gagatgtggg aggttttttt
     3181 aagcaagtaa aacctctaca aatgtggtaa aatcgaattt taacaaaata ttaacgctta
     3241 caatttcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg
     3301 cggatctgcg cagcaccatg gcctgaaata acctctgaaa gaggaacttg gttaggtacc
     3361 ttctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     3421 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     3481 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3541 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     3601 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     3661 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     3721 aagctcgatt cttctgacac tagcgccacc atgaagaagc ccgaactcac cgctaccagc
     3781 gttgaaaaat ttctcatcga gaagttcgac agtgtgagcg acctgatgca gttgtcggag
     3841 ggcgaagaga gccgagcctt cagcttcgat gtcggcggac gcggctatgt actgcgggtg
     3901 aatagctgcg ctgatggctt ctacaaagac cgctacgtgt accgccactt cgccagcgct
     3961 gcactaccca tccccgaagt gttggacatc ggcgagttca gcgagagcct gacatactgc
     4021 atcagtagac gcgcccaagg cgttactctc caagacctcc ccgaaacaga gctgcctgct
     4081 gtgttacagc ctgtcgccga agctatggat gctattgccg ccgccgacct cagtcaaacc
     4141 agcggcttcg gcccattcgg gccccaaggc atcggccagt acacaacctg gcgggatttc
     4201 atttgcgcca ttgctgatcc ccatgtctac cactggcaga ccgtgatgga cgacaccgtg
     4261 tccgccagcg tagctcaagc cctggacgaa ctgatgctgt gggccgaaga ctgtcccgag
     4321 gtgcgccacc tcgtccatgc cgacttcggc agcaacaacg tcctgaccga caacggccgc
     4381 atcaccgccg taatcgactg gtccgaagct atgttcgggg acagtcagta cgaggtggcc
     4441 aacatcttct tctggcggcc ctggctggct tgcatggagc agcagactcg ctacttcgag
     4501 cgccggcatc ccgagctggc cggcagccct cgtctgcgag cctacatgct gcgcatcggc
     4561 ctggatcagc tctaccagag cctcgtggac ggcaacttcg acgatgctgc ctgggctcaa
     4621 ggccgctgcg atgccatcgt ccgcagcggg gccggcaccg tcggtcgcac acaaatcgct
     4681 cgccggagcg cagccgtatg gaccgacggc tgcgtcgagg tgctggccga cagcggcaac
     4741 cgccggccca gtacacgacc gcgcgctaag gaggtaggtc gagtttagac tctagccatc
     4801 acgatggccg caataaaata tctttatttt cattacatct gtgtgttggt tttttgtgtg
     4861 aatcgatagc gataaggatc ctctttgcgc ttgcgttttc ccttgtccag atagcccagt
     4921 agctgacatt catccggggt cagcaccgtt tctgcggact ggctttctac gtaatggttt
     4981 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     5041 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     5101 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     5161 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     5221 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     5281 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttc aaagttctgc
     5341 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     5401 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     5461 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     5521 acttacttct gacaactatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     5581 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     5641 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     5701 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     5761 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     5821 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     5881 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     5941 agatcgctga gataggtgcc tcactgatta agcattggta attcgaaatg accgaccaag
     6001 cgacgcccaa ccggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     6061 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     6121 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     6181 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     6241 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     6301 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     6361 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     6421 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     6481 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     6541 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
     6601 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     6661 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     6721 atttcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     6781 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttata
     6841 gtccggaaat acaggaacgc acgctggatg gcccttcgct gggatggtga aaccatgaaa
     6901 aatggcagct tcagtggatt aagtgggggt aatgtggcct gtaccctctg gttgcatagg
     6961 tattcatacg gttaaaattt atcaggcgcg attgcggcag tttttcgggt ggtttgttgc
     7021 catttttacc tgtctgctgc cgtgatcgcg ctgaacgcgt tttagcggtg cgtacaatta
     7081 agggattatg gtaaatccac ttactgtctg ccctcgtagc catcgagata aaccgcagta
     7141 ctccggccac gatgcgtccg gcgtagagga tcgagatct
//
