LOCUS       sequence-426013-        7710 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of full-length human EIF2D. C-terminal NanoLuc 
            luciferase tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7710)
  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 7710)
  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_EIF2D:1-584
FEATURES             Location/Qualifiers
     source          1..7710
                     /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             2885..3397
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     primer_bind     complement(3473..3491)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    3528..3649
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3539..3558)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3593..3612
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3894..4251
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3894..4085
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3917..3937)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4102..4237
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4282..5319
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             5298..5306
                     /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    5343..5391
                     /note="synthetic polyadenylation signal"
     promoter        5547..5651
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5652..5720,5721..6512)
                     /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(5870..5889)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(6670..7258)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6750..6769)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    7374..7657
                     /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 caccatgttt gccaaggcct ttcgggtcaa gtccaacacg gccatcaagg ggtcggacag
     1141 gagaaagctt cgagctgatg tgacaactgc tttccccacc cttggaactg atcaagtctc
     1201 tgagttagta cctggaaagg aggagctcaa cattgtgaag ttgtatgctc acaaagggga
     1261 tgcagtgact gtgtacgtga gtggtggtaa ccccatcctc tttgaactgg agaaaaatct
     1321 gtatccaaca gtgtacacgc tgtggtccta tcctgatctt ctgccaacct ttacaacatg
     1381 gcctctggtg ctcgagaaac tggtaggggg agcagatttg atgctgcctg gactggtgat
     1441 gccccctgct ggtctgcctc aggtacagaa gggcgacctc tgtgccattt ctttggtggg
     1501 gaacagagcc cctgtagcca ttggagttgc agccatgtcc acagctgaga tgctcacgtc
     1561 aggcctgaag ggaaggggct tctctgtgct ccacacttac caggaccact tgtggcggtc
     1621 tggaaacaag tcctctccac cttccattgc tccactggcc ctggattcag cagatctcag
     1681 tgaagagaag gggtctgtcc agatggactc caccctgcag ggagacatga ggcacatgac
     1741 cctggagggg gaagaggaga atggggaggt tcaccaggca cgtgaagaca agtctctctc
     1801 agaagcccca gaagacacca gcaccagggg cctgaaccaa gactccacag atagcaaaac
     1861 gcttcaagaa caaatggatg agctgttaca gcaatgcttc ttacatgcct tgaagtgccg
     1921 agtcaaaaag gctgacctcc ctttactcac cagcactttc cttggcagcc acatgttctc
     1981 ctgctgcccc gaaggacgac aactggacat aaagaagtca agctacaaaa agctctctaa
     2041 gttcctgcag caaatgcagc aggagcagat tatacaggtg aaggagctga gcaaaggggt
     2101 ggagagcatt gtggctgtgg actggaaaca cccgaggatt acatctttcg tcatacccga
     2161 gccctccccg acctcccaga ctatccagga gggtagcagg gaacagccct atcaccctcc
     2221 agatataaaa cccctctact gtgtcccagc cagcatgacc ctgctcttcc aggagtctgg
     2281 ccacaagaag gggagctttc tggagggcag tgaggtccga acgatcgtca ttaactacgc
     2341 caagaaaaat gacctggttg atgcagacaa caaaaatctt gtgagattgg atcccatcct
     2401 atgtgactgc atcttagaga aaaatgaaca gcatacagtc atgaagcttc catgggacag
     2461 tcttctgacc aggtgtttgg aaaaattaca gcctgcctat caagtgaccc ttcccggaca
     2521 agagcccatt gtgaagaaag ggagaatctg tccaattgac atcaccctag cacaaagagc
     2581 gtctaataaa aaggtgaccg tggtccggaa cttggaggcc tatggtctgg acccatactc
     2641 agtggctgcc atccttcagc agcgatgcca ggctagcacc accgtcaatc ctgcccctgg
     2701 ggccaaggac agccttcagg tgcagatcca gggaaaccag gtccaccacc tcggctggct
     2761 attgcttgaa gagtatcagc tccctcgaaa acacatccaa ggtctagaaa aggccctcaa
     2821 acctggcaag aagaagtcta gatttgggcc caattcctgc aggcgagctc tcggctcgag
     2881 cggcgtcttc acactcgaag atttcgttgg ggactggcga cagacagccg gctacaacct
     2941 ggaccaagtc cttgaacagg gaggtgtgtc cagtttgttt cagaatctcg gggtgtccgt
     3001 aactccgatc caaaggattg tcctgagcgg tgaaaatggg ctgaagatcg acatccatgt
     3061 catcatcccg tatgaaggtc tgagcggcga ccaaatgggc cagatcgaaa aaatttttaa
     3121 ggtggtgtac cctgtggatg atcatcactt taaggtgatc ctgcactatg gcacactggt
     3181 aatcgacggg gttacgccga acatgatcga ctatttcgga cggccgtatg aaggcatcgc
     3241 cgtgttcgac ggcaaaaaga tcactgtaac agggaccctg tggaacggca acaaaattat
     3301 cgacgagcgc ctgatcaacc ccgacggctc cctgctgttc cgagtaacca tcaacggagt
     3361 gaccggctgg cggctgtgcg aacgcattct ggcgtaaggc cgcgactcta gggcatgcaa
     3421 gctgatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
     3481 caataactag cataacccct tggggcggcc gcttcgagca gacatgataa gatacattga
     3541 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     3601 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa
     3661 ttgcattcat tttatgtttc aggttcaggg ggagatgtgg gaggtttttt taagcaagta
     3721 aaacctctac aaatgtggta aaatcgaatt ttaacaaaat attaacgctt acaatttcct
     3781 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gcggatctgc
     3841 gcagcaccat ggcctgaaat aacctctgaa agaggaactt ggttaggtac cttctgaggc
     3901 ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     3961 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     4021 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     4081 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     4141 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     4201 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcgat
     4261 tcttctgaca ctagcgccac catgaagaag cccgaactca ccgctaccag cgttgaaaaa
     4321 tttctcatcg agaagttcga cagtgtgagc gacctgatgc agttgtcgga gggcgaagag
     4381 agccgagcct tcagcttcga tgtcggcgga cgcggctatg tactgcgggt gaatagctgc
     4441 gctgatggct tctacaaaga ccgctacgtg taccgccact tcgccagcgc tgcactaccc
     4501 atccccgaag tgttggacat cggcgagttc agcgagagcc tgacatactg catcagtaga
     4561 cgcgcccaag gcgttactct ccaagacctc cccgaaacag agctgcctgc tgtgttacag
     4621 cctgtcgccg aagctatgga tgctattgcc gccgccgacc tcagtcaaac cagcggcttc
     4681 ggcccattcg ggccccaagg catcggccag tacacaacct ggcgggattt catttgcgcc
     4741 attgctgatc cccatgtcta ccactggcag accgtgatgg acgacaccgt gtccgccagc
     4801 gtagctcaag ccctggacga actgatgctg tgggccgaag actgtcccga ggtgcgccac
     4861 ctcgtccatg ccgacttcgg cagcaacaac gtcctgaccg acaacggccg catcaccgcc
     4921 gtaatcgact ggtccgaagc tatgttcggg gacagtcagt acgaggtggc caacatcttc
     4981 ttctggcggc cctggctggc ttgcatggag cagcagactc gctacttcga gcgccggcat
     5041 cccgagctgg ccggcagccc tcgtctgcga gcctacatgc tgcgcatcgg cctggatcag
     5101 ctctaccaga gcctcgtgga cggcaacttc gacgatgctg cctgggctca aggccgctgc
     5161 gatgccatcg tccgcagcgg ggccggcacc gtcggtcgca cacaaatcgc tcgccggagc
     5221 gcagccgtat ggaccgacgg ctgcgtcgag gtgctggccg acagcggcaa ccgccggccc
     5281 agtacacgac cgcgcgctaa ggaggtaggt cgagtttaga ctctagccat cacgatggcc
     5341 gcaataaaat atctttattt tcattacatc tgtgtgttgg ttttttgtgt gaatcgatag
     5401 cgataaggat cctctttgcg cttgcgtttt cccttgtcca gatagcccag tagctgacat
     5461 tcatccgggg tcagcaccgt ttctgcggac tggctttcta cgtaatggtt tcttagacgt
     5521 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     5581 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     5641 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     5701 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     5761 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     5821 gttttcgccc cgaagaacgt tttccaatga tgagcacttt caaagttctg ctatgtggcg
     5881 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     5941 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     6001 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     6061 tgacaactat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     6121 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     6181 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     6241 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     6301 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     6361 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     6421 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     6481 agataggtgc ctcactgatt aagcattggt aattcgaaat gaccgaccaa gcgacgccca
     6541 accggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     6601 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     6661 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     6721 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     6781 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     6841 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     6901 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     6961 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     7021 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     7081 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
     7141 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     7201 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatttcaaga
     7261 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     7321 gattttggtc atgagattat caaaaaggat cttcacctag atccttttat agtccggaaa
     7381 tacaggaacg cacgctggat ggcccttcgc tgggatggtg aaaccatgaa aaatggcagc
     7441 ttcagtggat taagtggggg taatgtggcc tgtaccctct ggttgcatag gtattcatac
     7501 ggttaaaatt tatcaggcgc gattgcggca gtttttcggg tggtttgttg ccatttttac
     7561 ctgtctgctg ccgtgatcgc gctgaacgcg ttttagcggt gcgtacaatt aagggattat
     7621 ggtaaatcca cttactgtct gccctcgtag ccatcgagat aaaccgcagt actccggcca
     7681 cgatgcgtcc ggcgtagagg atcgagatct
//
