LOCUS       sequence-425957-        6822 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of full-length human SKIC8. N-terminal NanoLuc 
            luciferase tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6822)
  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 6822)
  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_SKIC8:1-305
FEATURES             Location/Qualifiers
     source          1..6822
                     /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(2590..2608)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    2640..2761
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2651..2670)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2705..2724
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3006..3363
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3006..3197
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3029..3049)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3214..3349
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3394..4431
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             4410..4418
                     /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    4455..4503
                     /note="synthetic polyadenylation signal"
     promoter        4659..4763
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4764..4832,4833..5624)
                     /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(4982..5001)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(5782..6370)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5862..5881)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    6486..6769
                     /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 accaaccagt
     1621 acggtattct cttcaaacaa gagcaagccc atgatgatgc catttggtca gttgcttggg
     1681 ggacaaacaa gaaggaaaac tctgagacag tggtcacagg ctccctagat gacctggtga
     1741 aggtctggaa atggcgtgat gagaggctgg acctacagtg gagtctggag ggacatcagc
     1801 tgggagtggt gtctgtggac atcagccaca ccctgcccat tgctgcatcc agctctcttg
     1861 atgctcatat tcgtctttgg gacttggaaa atggcaaaca gataaagtcc atagatgcag
     1921 gacctgtgga tgcctggact ttggcctttt ctcctgattc ccagtatctg gccacaggaa
     1981 ctcatgtcgg gaaagtgaac atttttggtg tggaaagtgg gaaaaaggaa tattctttgg
     2041 acacgagagg aaaattcatt cttagtattg catatagtcc tgatgggaaa tacctagcca
     2101 gtggagccat agatggaatc atcaatattt ttgatattgc aactggaaaa cttctgcata
     2161 ccctggaagg ccatgccatg cccattcgct ccttgacctt ttccccggac tcccagctcc
     2221 ttgtcactgc ttcagatgat ggctacatca agatctatga tgtacaacat gccaatttgg
     2281 ctggcacgct gagcggccat gcctcctggg tgctgaacgt tgcattctgt cctgatgaca
     2341 ctcactttgt ttccagttcg tctgacaaaa gtgtaaaagt ttgggatgtt ggaacgagga
     2401 cttgtgttca caccttcttt gatcaccagg atcaggtctg gggagtaaaa tacaatggaa
     2461 atggttcaaa aattgtgtct gttggagatg accaggaaat tcacatctat gattgtccaa
     2521 tttaataatc tagatttggg cccaattcct gcaggatttt gcggccgctt gctgagttgg
     2581 ctgctgccac cgctgagcaa taactagcat aaccccttgg ccgcttcgag cagacatgat
     2641 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
     2701 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     2761 taacaacaac aattgcattc attttatgtt tcaggttcag ggggagatgt gggaggtttt
     2821 tttaagcaag taaaacctct acaaatgtgg taaaatcgaa ttttaacaaa atattaacgc
     2881 ttacaatttc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     2941 acgcggatct gcgcagcacc atggcctgaa ataacctctg aaagaggaac ttggttaggt
     3001 accttctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     3061 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     3121 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3181 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     3241 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     3301 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     3361 aaaaagctcg attcttctga cactagcgcc accatgaaga agcccgaact caccgctacc
     3421 agcgttgaaa aatttctcat cgagaagttc gacagtgtga gcgacctgat gcagttgtcg
     3481 gagggcgaag agagccgagc cttcagcttc gatgtcggcg gacgcggcta tgtactgcgg
     3541 gtgaatagct gcgctgatgg cttctacaaa gaccgctacg tgtaccgcca cttcgccagc
     3601 gctgcactac ccatccccga agtgttggac atcggcgagt tcagcgagag cctgacatac
     3661 tgcatcagta gacgcgccca aggcgttact ctccaagacc tccccgaaac agagctgcct
     3721 gctgtgttac agcctgtcgc cgaagctatg gatgctattg ccgccgccga cctcagtcaa
     3781 accagcggct tcggcccatt cgggccccaa ggcatcggcc agtacacaac ctggcgggat
     3841 ttcatttgcg ccattgctga tccccatgtc taccactggc agaccgtgat ggacgacacc
     3901 gtgtccgcca gcgtagctca agccctggac gaactgatgc tgtgggccga agactgtccc
     3961 gaggtgcgcc acctcgtcca tgccgacttc ggcagcaaca acgtcctgac cgacaacggc
     4021 cgcatcaccg ccgtaatcga ctggtccgaa gctatgttcg gggacagtca gtacgaggtg
     4081 gccaacatct tcttctggcg gccctggctg gcttgcatgg agcagcagac tcgctacttc
     4141 gagcgccggc atcccgagct ggccggcagc cctcgtctgc gagcctacat gctgcgcatc
     4201 ggcctggatc agctctacca gagcctcgtg gacggcaact tcgacgatgc tgcctgggct
     4261 caaggccgct gcgatgccat cgtccgcagc ggggccggca ccgtcggtcg cacacaaatc
     4321 gctcgccgga gcgcagccgt atggaccgac ggctgcgtcg aggtgctggc cgacagcggc
     4381 aaccgccggc ccagtacacg accgcgcgct aaggaggtag gtcgagttta gactctagcc
     4441 atcacgatgg ccgcaataaa atatctttat tttcattaca tctgtgtgtt ggttttttgt
     4501 gtgaatcgat agcgataagg atcctctttg cgcttgcgtt ttcccttgtc cagatagccc
     4561 agtagctgac attcatccgg ggtcagcacc gtttctgcgg actggctttc tacgtaatgg
     4621 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     4681 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     4741 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     4801 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     4861 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     4921 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact ttcaaagttc
     4981 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     5041 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     5101 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     5161 ccaacttact tctgacaact atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     5221 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     5281 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     5341 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     5401 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     5461 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     5521 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     5581 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaattcgaa atgaccgacc
     5641 aagcgacgcc caaccggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     5701 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     5761 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     5821 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     5881 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     5941 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     6001 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     6061 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     6121 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     6181 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
     6241 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     6301 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     6361 aggatttcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     6421 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     6481 atagtccgga aatacaggaa cgcacgctgg atggcccttc gctgggatgg tgaaaccatg
     6541 aaaaatggca gcttcagtgg attaagtggg ggtaatgtgg cctgtaccct ctggttgcat
     6601 aggtattcat acggttaaaa tttatcaggc gcgattgcgg cagtttttcg ggtggtttgt
     6661 tgccattttt acctgtctgc tgccgtgatc gcgctgaacg cgttttagcg gtgcgtacaa
     6721 ttaagggatt atggtaaatc cacttactgt ctgccctcgt agccatcgag ataaaccgca
     6781 gtactccggc cacgatgcgt ccggcgtaga ggatcgagat ct
//
