LOCUS       sequence-423285-        6807 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of full-length human GID4. N-terminal NanoLuc 
            luciferase tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6807)
  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 6807)
  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-N_GID4:1-300
FEATURES             Location/Qualifiers
     source          1..6807
                     /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             1065..1577
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     primer_bind     complement(2575..2593)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    2625..2746
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2636..2655)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2690..2709
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        2991..3348
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2991..3182
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3014..3034)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3199..3334
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3379..4416
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             4395..4403
                     /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    4440..4488
                     /note="synthetic polyadenylation signal"
     promoter        4644..4748
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4749..4817,4818..5609)
                     /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(4967..4986)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(5767..6355)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5847..5866)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    6471..6754
                     /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 ggctagcgct caccatggtc ttcacactcg
     1081 aagatttcgt tggggactgg cgacagacag ccggctacaa cctggaccaa gtccttgaac
     1141 agggaggtgt gtccagtttg tttcagaatc tcggggtgtc cgtaactccg atccaaagga
     1201 ttgtcctgag cggtgaaaat gggctgaaga tcgacatcca tgtcatcatc ccgtatgaag
     1261 gtctgagcgg cgaccaaatg ggccagatcg aaaaaatttt taaggtggtg taccctgtgg
     1321 atgatcatca ctttaaggtg atcctgcact atggcacact ggtaatcgac ggggttacgc
     1381 cgaacatgat cgactatttc ggacggccgt atgaaggcat cgccgtgttc gacggcaaaa
     1441 agatcactgt aacagggacc ctgtggaacg gcaacaaaat tatcgacgag cgcctgatca
     1501 accccgacgg ctccctgctg ttccgagtaa ccatcaacgg agtgaccggc tggcggctgt
     1561 gcgaacgcat tctggcgggc tcgagcggcg cgatcgcttc cgaattcatg tgtgcgcgag
     1621 ggcaagtcgg gagggggacc cagctcagga ctgggaggcc ctgctcgcag gtccctgggt
     1681 cccggtggcg gccggagcgc ttgctccgca ggcagcgggc gggtggtcgc ccctcccgcc
     1741 cccaccccgc gcgtgcgcgc cccggcctct ccctccccgc caccctcctc ggctcccgcg
     1801 cggcggcggc ggttcctctc ccactccccc cagccctggc tccgggggac cccgcgatgc
     1861 cggtccgcac cgagtgtccc ccgccggccg gtgcctccgc tgcctccgcg gcctcactca
     1921 tcccgccgcc gcccatcaac acccagcagc ccggcgtggc caccagcctg ctctacagcg
     1981 gctccaagtt ccgcggccac cagaagagca aggggaactc gtacgacgta gaggtggtgc
     2041 tgcagcacgt ggacacggga aactcttacc tttgtgggta cttgaagatt aaaggcctta
     2101 ctgaggagta tccaaccctt acaaccttct tcgaaggaga aataatcagc aaaaaacacc
     2161 ctttcttaac tcgcaagtgg gatgcagatg aagatgttga tcggaaacac tggggcaagt
     2221 ttctggcttt ttatcagtat gcaaaatcat ttaactcaga tgactttgat tatgaagagc
     2281 tgaagaatgg agactacgtc ttcatgaggt ggaaggaaca gtttctggtc ccagatcaca
     2341 cgatcaaaga catcagtggt gcttcttttg ccgggttcta ctacatctgc tttcagaagt
     2401 cagcagcctc catagagggc tactactacc ataggagttc agaatggtat cagtccctca
     2461 atctaaccca tgttcctgaa cacagtgcac ccatctatga attccggtaa taatctagat
     2521 ttgggcccaa ttcctgcagg attttgcggc cgcttgctga gttggctgct gccaccgctg
     2581 agcaataact agcataaccc cttggccgct tcgagcagac atgataagat acattgatga
     2641 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
     2701 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg
     2761 cattcatttt atgtttcagg ttcaggggga gatgtgggag gtttttttaa gcaagtaaaa
     2821 cctctacaaa tgtggtaaaa tcgaatttta acaaaatatt aacgcttaca atttcctgat
     2881 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgcg gatctgcgca
     2941 gcaccatggc ctgaaataac ctctgaaaga ggaacttggt taggtacctt ctgaggcgga
     3001 aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     3061 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     3121 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     3181 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     3241 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     3301 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcgattct
     3361 tctgacacta gcgccaccat gaagaagccc gaactcaccg ctaccagcgt tgaaaaattt
     3421 ctcatcgaga agttcgacag tgtgagcgac ctgatgcagt tgtcggaggg cgaagagagc
     3481 cgagccttca gcttcgatgt cggcggacgc ggctatgtac tgcgggtgaa tagctgcgct
     3541 gatggcttct acaaagaccg ctacgtgtac cgccacttcg ccagcgctgc actacccatc
     3601 cccgaagtgt tggacatcgg cgagttcagc gagagcctga catactgcat cagtagacgc
     3661 gcccaaggcg ttactctcca agacctcccc gaaacagagc tgcctgctgt gttacagcct
     3721 gtcgccgaag ctatggatgc tattgccgcc gccgacctca gtcaaaccag cggcttcggc
     3781 ccattcgggc cccaaggcat cggccagtac acaacctggc gggatttcat ttgcgccatt
     3841 gctgatcccc atgtctacca ctggcagacc gtgatggacg acaccgtgtc cgccagcgta
     3901 gctcaagccc tggacgaact gatgctgtgg gccgaagact gtcccgaggt gcgccacctc
     3961 gtccatgccg acttcggcag caacaacgtc ctgaccgaca acggccgcat caccgccgta
     4021 atcgactggt ccgaagctat gttcggggac agtcagtacg aggtggccaa catcttcttc
     4081 tggcggccct ggctggcttg catggagcag cagactcgct acttcgagcg ccggcatccc
     4141 gagctggccg gcagccctcg tctgcgagcc tacatgctgc gcatcggcct ggatcagctc
     4201 taccagagcc tcgtggacgg caacttcgac gatgctgcct gggctcaagg ccgctgcgat
     4261 gccatcgtcc gcagcggggc cggcaccgtc ggtcgcacac aaatcgctcg ccggagcgca
     4321 gccgtatgga ccgacggctg cgtcgaggtg ctggccgaca gcggcaaccg ccggcccagt
     4381 acacgaccgc gcgctaagga ggtaggtcga gtttagactc tagccatcac gatggccgca
     4441 ataaaatatc tttattttca ttacatctgt gtgttggttt tttgtgtgaa tcgatagcga
     4501 taaggatcct ctttgcgctt gcgttttccc ttgtccagat agcccagtag ctgacattca
     4561 tccggggtca gcaccgtttc tgcggactgg ctttctacgt aatggtttct tagacgtcag
     4621 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     4681 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     4741 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     4801 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     4861 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     4921 ttcgccccga agaacgtttt ccaatgatga gcactttcaa agttctgcta tgtggcgcgg
     4981 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     5041 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     5101 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     5161 caactatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     5221 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     5281 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     5341 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     5401 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     5461 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     5521 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     5581 taggtgcctc actgattaag cattggtaat tcgaaatgac cgaccaagcg acgcccaacc
     5641 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     5701 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     5761 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     5821 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     5881 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     5941 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     6001 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     6061 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     6121 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     6181 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
     6241 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     6301 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ttcaagaaga
     6361 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     6421 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttatagt ccggaaatac
     6481 aggaacgcac gctggatggc ccttcgctgg gatggtgaaa ccatgaaaaa tggcagcttc
     6541 agtggattaa gtgggggtaa tgtggcctgt accctctggt tgcataggta ttcatacggt
     6601 taaaatttat caggcgcgat tgcggcagtt tttcgggtgg tttgttgcca tttttacctg
     6661 tctgctgccg tgatcgcgct gaacgcgttt tagcggtgcg tacaattaag ggattatggt
     6721 aaatccactt actgtctgcc ctcgtagcca tcgagataaa ccgcagtact ccggccacga
     6781 tgcgtccggc gtagaggatc gagatct
//
