LOCUS       sequence-425958-        6873 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of full-length human SKIC8. C-terminal NanoLuc 
            luciferase tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6873)
  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 6873)
  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_SKIC8:1-305
FEATURES             Location/Qualifiers
     source          1..6873
                     /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             2048..2560
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     primer_bind     complement(2636..2654)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    2691..2812
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2702..2721)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2756..2775
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3057..3414
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3057..3248
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3080..3100)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3265..3400
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3445..4482
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             4461..4469
                     /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    4506..4554
                     /note="synthetic polyadenylation signal"
     promoter        4710..4814
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4815..4883,4884..5675)
                     /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(5033..5052)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(5833..6421)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5913..5932)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    6537..6820
                     /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 caccatgacc aaccagtacg gtattctctt caaacaagag caagcccatg atgatgccat
     1141 ttggtcagtt gcttggggga caaacaagaa ggaaaactct gagacagtgg tcacaggctc
     1201 cctagatgac ctggtgaagg tctggaaatg gcgtgatgag aggctggacc tacagtggag
     1261 tctggaggga catcagctgg gagtggtgtc tgtggacatc agccacaccc tgcccattgc
     1321 tgcatccagc tctcttgatg ctcatattcg tctttgggac ttggaaaatg gcaaacagat
     1381 aaagtccata gatgcaggac ctgtggatgc ctggactttg gccttttctc ctgattccca
     1441 gtatctggcc acaggaactc atgtcgggaa agtgaacatt tttggtgtgg aaagtgggaa
     1501 aaaggaatat tctttggaca cgagaggaaa attcattctt agtattgcat atagtcctga
     1561 tgggaaatac ctagccagtg gagccataga tggaatcatc aatatttttg atattgcaac
     1621 tggaaaactt ctgcataccc tggaaggcca tgccatgccc attcgctcct tgaccttttc
     1681 cccggactcc cagctccttg tcactgcttc agatgatggc tacatcaaga tctatgatgt
     1741 acaacatgcc aatttggctg gcacgctgag cggccatgcc tcctgggtgc tgaacgttgc
     1801 attctgtcct gatgacactc actttgtttc cagttcgtct gacaaaagtg taaaagtttg
     1861 ggatgttgga acgaggactt gtgttcacac cttctttgat caccaggatc aggtctgggg
     1921 agtaaaatac aatggaaatg gttcaaaaat tgtgtctgtt ggagatgacc aggaaattca
     1981 catctatgat tgtccaattt ctagatttgg gcccaattcc tgcaggcgag ctctcggctc
     2041 gagcggcgtc ttcacactcg aagatttcgt tggggactgg cgacagacag ccggctacaa
     2101 cctggaccaa gtccttgaac agggaggtgt gtccagtttg tttcagaatc tcggggtgtc
     2161 cgtaactccg atccaaagga ttgtcctgag cggtgaaaat gggctgaaga tcgacatcca
     2221 tgtcatcatc ccgtatgaag gtctgagcgg cgaccaaatg ggccagatcg aaaaaatttt
     2281 taaggtggtg taccctgtgg atgatcatca ctttaaggtg atcctgcact atggcacact
     2341 ggtaatcgac ggggttacgc cgaacatgat cgactatttc ggacggccgt atgaaggcat
     2401 cgccgtgttc gacggcaaaa agatcactgt aacagggacc ctgtggaacg gcaacaaaat
     2461 tatcgacgag cgcctgatca accccgacgg ctccctgctg ttccgagtaa ccatcaacgg
     2521 agtgaccggc tggcggctgt gcgaacgcat tctggcgtaa ggccgcgact ctagggcatg
     2581 caagctgatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     2641 gagcaataac tagcataacc ccttggggcg gccgcttcga gcagacatga taagatacat
     2701 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
     2761 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa
     2821 caattgcatt cattttatgt ttcaggttca gggggagatg tgggaggttt ttttaagcaa
     2881 gtaaaacctc tacaaatgtg gtaaaatcga attttaacaa aatattaacg cttacaattt
     2941 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgcggatc
     3001 tgcgcagcac catggcctga aataacctct gaaagaggaa cttggttagg taccttctga
     3061 ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     3121 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     3181 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3241 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     3301 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     3361 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     3421 gattcttctg acactagcgc caccatgaag aagcccgaac tcaccgctac cagcgttgaa
     3481 aaatttctca tcgagaagtt cgacagtgtg agcgacctga tgcagttgtc ggagggcgaa
     3541 gagagccgag ccttcagctt cgatgtcggc ggacgcggct atgtactgcg ggtgaatagc
     3601 tgcgctgatg gcttctacaa agaccgctac gtgtaccgcc acttcgccag cgctgcacta
     3661 cccatccccg aagtgttgga catcggcgag ttcagcgaga gcctgacata ctgcatcagt
     3721 agacgcgccc aaggcgttac tctccaagac ctccccgaaa cagagctgcc tgctgtgtta
     3781 cagcctgtcg ccgaagctat ggatgctatt gccgccgccg acctcagtca aaccagcggc
     3841 ttcggcccat tcgggcccca aggcatcggc cagtacacaa cctggcggga tttcatttgc
     3901 gccattgctg atccccatgt ctaccactgg cagaccgtga tggacgacac cgtgtccgcc
     3961 agcgtagctc aagccctgga cgaactgatg ctgtgggccg aagactgtcc cgaggtgcgc
     4021 cacctcgtcc atgccgactt cggcagcaac aacgtcctga ccgacaacgg ccgcatcacc
     4081 gccgtaatcg actggtccga agctatgttc ggggacagtc agtacgaggt ggccaacatc
     4141 ttcttctggc ggccctggct ggcttgcatg gagcagcaga ctcgctactt cgagcgccgg
     4201 catcccgagc tggccggcag ccctcgtctg cgagcctaca tgctgcgcat cggcctggat
     4261 cagctctacc agagcctcgt ggacggcaac ttcgacgatg ctgcctgggc tcaaggccgc
     4321 tgcgatgcca tcgtccgcag cggggccggc accgtcggtc gcacacaaat cgctcgccgg
     4381 agcgcagccg tatggaccga cggctgcgtc gaggtgctgg ccgacagcgg caaccgccgg
     4441 cccagtacac gaccgcgcgc taaggaggta ggtcgagttt agactctagc catcacgatg
     4501 gccgcaataa aatatcttta ttttcattac atctgtgtgt tggttttttg tgtgaatcga
     4561 tagcgataag gatcctcttt gcgcttgcgt tttcccttgt ccagatagcc cagtagctga
     4621 cattcatccg gggtcagcac cgtttctgcg gactggcttt ctacgtaatg gtttcttaga
     4681 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     4741 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     4801 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     4861 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     4921 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     4981 agagttttcg ccccgaagaa cgttttccaa tgatgagcac tttcaaagtt ctgctatgtg
     5041 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5101 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     5161 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5221 ttctgacaac tatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     5281 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     5341 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     5401 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     5461 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     5521 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     5581 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     5641 ctgagatagg tgcctcactg attaagcatt ggtaattcga aatgaccgac caagcgacgc
     5701 ccaaccggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     5761 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     5821 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     5881 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     5941 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     6001 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     6061 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     6121 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     6181 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     6241 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct
     6301 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     6361 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatttca
     6421 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     6481 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt tatagtccgg
     6541 aaatacagga acgcacgctg gatggccctt cgctgggatg gtgaaaccat gaaaaatggc
     6601 agcttcagtg gattaagtgg gggtaatgtg gcctgtaccc tctggttgca taggtattca
     6661 tacggttaaa atttatcagg cgcgattgcg gcagtttttc gggtggtttg ttgccatttt
     6721 tacctgtctg ctgccgtgat cgcgctgaac gcgttttagc ggtgcgtaca attaagggat
     6781 tatggtaaat ccacttactg tctgccctcg tagccatcga gataaaccgc agtactccgg
     6841 ccacgatgcg tccggcgtag aggatcgaga tct
//
