LOCUS       sequence-423289-        6858 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of full-length human GID4. C-terminal NanoLuc 
            luciferase tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6858)
  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 6858)
  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_GID4:1-300
FEATURES             Location/Qualifiers
     source          1..6858
                     /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             2033..2545
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     primer_bind     complement(2621..2639)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    2676..2797
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2687..2706)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2741..2760
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3042..3399
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3042..3233
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3065..3085)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3250..3385
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3430..4467
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             4446..4454
                     /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    4491..4539
                     /note="synthetic polyadenylation signal"
     promoter        4695..4799
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4800..4868,4869..5660)
                     /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(5018..5037)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(5818..6406)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5898..5917)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    6522..6805
                     /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 caccatgtgt gcgcgagggc aagtcgggag ggggacccag ctcaggactg ggaggccctg
     1141 ctcgcaggtc cctgggtccc ggtggcggcc ggagcgcttg ctccgcaggc agcgggcggg
     1201 tggtcgcccc tcccgccccc accccgcgcg tgcgcgcccc ggcctctccc tccccgccac
     1261 cctcctcggc tcccgcgcgg cggcggcggt tcctctccca ctccccccag ccctggctcc
     1321 gggggacccc gcgatgccgg tccgcaccga gtgtcccccg ccggccggtg cctccgctgc
     1381 ctccgcggcc tcactcatcc cgccgccgcc catcaacacc cagcagcccg gcgtggccac
     1441 cagcctgctc tacagcggct ccaagttccg cggccaccag aagagcaagg ggaactcgta
     1501 cgacgtagag gtggtgctgc agcacgtgga cacgggaaac tcttaccttt gtgggtactt
     1561 gaagattaaa ggccttactg aggagtatcc aacccttaca accttcttcg aaggagaaat
     1621 aatcagcaaa aaacaccctt tcttaactcg caagtgggat gcagatgaag atgttgatcg
     1681 gaaacactgg ggcaagtttc tggcttttta tcagtatgca aaatcattta actcagatga
     1741 ctttgattat gaagagctga agaatggaga ctacgtcttc atgaggtgga aggaacagtt
     1801 tctggtccca gatcacacga tcaaagacat cagtggtgct tcttttgccg ggttctacta
     1861 catctgcttt cagaagtcag cagcctccat agagggctac tactaccata ggagttcaga
     1921 atggtatcag tccctcaatc taacccatgt tcctgaacac agtgcaccca tctatgaatt
     1981 ccggtctaga tttgggccca attcctgcag gcgagctctc ggctcgagcg gcgtcttcac
     2041 actcgaagat ttcgttgggg actggcgaca gacagccggc tacaacctgg accaagtcct
     2101 tgaacaggga ggtgtgtcca gtttgtttca gaatctcggg gtgtccgtaa ctccgatcca
     2161 aaggattgtc ctgagcggtg aaaatgggct gaagatcgac atccatgtca tcatcccgta
     2221 tgaaggtctg agcggcgacc aaatgggcca gatcgaaaaa atttttaagg tggtgtaccc
     2281 tgtggatgat catcacttta aggtgatcct gcactatggc acactggtaa tcgacggggt
     2341 tacgccgaac atgatcgact atttcggacg gccgtatgaa ggcatcgccg tgttcgacgg
     2401 caaaaagatc actgtaacag ggaccctgtg gaacggcaac aaaattatcg acgagcgcct
     2461 gatcaacccc gacggctccc tgctgttccg agtaaccatc aacggagtga ccggctggcg
     2521 gctgtgcgaa cgcattctgg cgtaaggccg cgactctagg gcatgcaagc tgatccggct
     2581 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
     2641 taaccccttg gggcggccgc ttcgagcaga catgataaga tacattgatg agtttggaca
     2701 aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc
     2761 tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt
     2821 tatgtttcag gttcaggggg agatgtggga ggttttttta agcaagtaaa acctctacaa
     2881 atgtggtaaa atcgaatttt aacaaaatat taacgcttac aatttcctga tgcggtattt
     2941 tctccttacg catctgtgcg gtatttcaca ccgcatacgc ggatctgcgc agcaccatgg
     3001 cctgaaataa cctctgaaag aggaacttgg ttaggtacct tctgaggcgg aaagaaccag
     3061 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     3121 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     3181 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     3241 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     3301 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
     3361 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcgattc ttctgacact
     3421 agcgccacca tgaagaagcc cgaactcacc gctaccagcg ttgaaaaatt tctcatcgag
     3481 aagttcgaca gtgtgagcga cctgatgcag ttgtcggagg gcgaagagag ccgagccttc
     3541 agcttcgatg tcggcggacg cggctatgta ctgcgggtga atagctgcgc tgatggcttc
     3601 tacaaagacc gctacgtgta ccgccacttc gccagcgctg cactacccat ccccgaagtg
     3661 ttggacatcg gcgagttcag cgagagcctg acatactgca tcagtagacg cgcccaaggc
     3721 gttactctcc aagacctccc cgaaacagag ctgcctgctg tgttacagcc tgtcgccgaa
     3781 gctatggatg ctattgccgc cgccgacctc agtcaaacca gcggcttcgg cccattcggg
     3841 ccccaaggca tcggccagta cacaacctgg cgggatttca tttgcgccat tgctgatccc
     3901 catgtctacc actggcagac cgtgatggac gacaccgtgt ccgccagcgt agctcaagcc
     3961 ctggacgaac tgatgctgtg ggccgaagac tgtcccgagg tgcgccacct cgtccatgcc
     4021 gacttcggca gcaacaacgt cctgaccgac aacggccgca tcaccgccgt aatcgactgg
     4081 tccgaagcta tgttcgggga cagtcagtac gaggtggcca acatcttctt ctggcggccc
     4141 tggctggctt gcatggagca gcagactcgc tacttcgagc gccggcatcc cgagctggcc
     4201 ggcagccctc gtctgcgagc ctacatgctg cgcatcggcc tggatcagct ctaccagagc
     4261 ctcgtggacg gcaacttcga cgatgctgcc tgggctcaag gccgctgcga tgccatcgtc
     4321 cgcagcgggg ccggcaccgt cggtcgcaca caaatcgctc gccggagcgc agccgtatgg
     4381 accgacggct gcgtcgaggt gctggccgac agcggcaacc gccggcccag tacacgaccg
     4441 cgcgctaagg aggtaggtcg agtttagact ctagccatca cgatggccgc aataaaatat
     4501 ctttattttc attacatctg tgtgttggtt ttttgtgtga atcgatagcg ataaggatcc
     4561 tctttgcgct tgcgttttcc cttgtccaga tagcccagta gctgacattc atccggggtc
     4621 agcaccgttt ctgcggactg gctttctacg taatggtttc ttagacgtca ggtggcactt
     4681 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     4741 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     4801 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     4861 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     4921 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     4981 aagaacgttt tccaatgatg agcactttca aagttctgct atgtggcgcg gtattatccc
     5041 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     5101 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     5161 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaactatcg
     5221 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     5281 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     5341 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     5401 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     5461 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     5521 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     5581 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     5641 cactgattaa gcattggtaa ttcgaaatga ccgaccaagc gacgcccaac cggtatcagc
     5701 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     5761 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     5821 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     5881 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     5941 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     6001 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     6061 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     6121 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     6181 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     6241 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     6301 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     6361 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tttcaagaag atcctttgat
     6421 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     6481 gagattatca aaaaggatct tcacctagat ccttttatag tccggaaata caggaacgca
     6541 cgctggatgg cccttcgctg ggatggtgaa accatgaaaa atggcagctt cagtggatta
     6601 agtgggggta atgtggcctg taccctctgg ttgcataggt attcatacgg ttaaaattta
     6661 tcaggcgcga ttgcggcagt ttttcgggtg gtttgttgcc atttttacct gtctgctgcc
     6721 gtgatcgcgc tgaacgcgtt ttagcggtgc gtacaattaa gggattatgg taaatccact
     6781 tactgtctgc cctcgtagcc atcgagataa accgcagtac tccggccacg atgcgtccgg
     6841 cgtagaggat cgagatct
//
