LOCUS       sequence-423300-        6264 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of full-length human DDA1. C-terminal NanoLuc 
            luciferase tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6264)
  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 6264)
  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_DDA1:1-102
FEATURES             Location/Qualifiers
     source          1..6264
                     /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"
     regulatory      1079..1088
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1439..1951
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     primer_bind     complement(2027..2045)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    2082..2203
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2093..2112)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2147..2166
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        2448..2805
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2448..2639
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2471..2491)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2656..2791
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2836..3873
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             3852..3860
                     /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    3897..3945
                     /note="synthetic polyadenylation signal"
     promoter        4101..4205
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4206..4274,4275..5066)
                     /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(4424..4443)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(5224..5812)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5304..5323)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    5928..6211
                     /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 caccatggca gattttttga aaggactgcc tgtctacaac aaaagcaatt ttagtcgatt
     1141 tcacgcggac tccgtgtgca aagcctcgaa ccgacggccc tcagtctacc tgcctacccg
     1201 cgagtacccg tctgaacaga tcatcgtgac agaaaagaca aacatcctcc tgcgctacct
     1261 gcatcagcaa tgggacaaaa agaacgctgc caagaagaga gaccaggagc aagtggagct
     1321 ggaaggcgag agctccgcac ctccccgcaa ggtggcgcgg accgacagcc cagacatgca
     1381 cgaggacact tctagatttg ggcccaattc ctgcaggcga gctctcggct cgagcggcgt
     1441 cttcacactc gaagatttcg ttggggactg gcgacagaca gccggctaca acctggacca
     1501 agtccttgaa cagggaggtg tgtccagttt gtttcagaat ctcggggtgt ccgtaactcc
     1561 gatccaaagg attgtcctga gcggtgaaaa tgggctgaag atcgacatcc atgtcatcat
     1621 cccgtatgaa ggtctgagcg gcgaccaaat gggccagatc gaaaaaattt ttaaggtggt
     1681 gtaccctgtg gatgatcatc actttaaggt gatcctgcac tatggcacac tggtaatcga
     1741 cggggttacg ccgaacatga tcgactattt cggacggccg tatgaaggca tcgccgtgtt
     1801 cgacggcaaa aagatcactg taacagggac cctgtggaac ggcaacaaaa ttatcgacga
     1861 gcgcctgatc aaccccgacg gctccctgct gttccgagta accatcaacg gagtgaccgg
     1921 ctggcggctg tgcgaacgca ttctggcgta aggccgcgac tctagggcat gcaagctgat
     1981 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     2041 ctagcataac cccttggggc ggccgcttcg agcagacatg ataagataca ttgatgagtt
     2101 tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc
     2161 tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat
     2221 tcattttatg tttcaggttc agggggagat gtgggaggtt tttttaagca agtaaaacct
     2281 ctacaaatgt ggtaaaatcg aattttaaca aaatattaac gcttacaatt tcctgatgcg
     2341 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgcggat ctgcgcagca
     2401 ccatggcctg aaataacctc tgaaagagga acttggttag gtaccttctg aggcggaaag
     2461 aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     2521 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     2581 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     2641 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     2701 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     2761 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cgattcttct
     2821 gacactagcg ccaccatgaa gaagcccgaa ctcaccgcta ccagcgttga aaaatttctc
     2881 atcgagaagt tcgacagtgt gagcgacctg atgcagttgt cggagggcga agagagccga
     2941 gccttcagct tcgatgtcgg cggacgcggc tatgtactgc gggtgaatag ctgcgctgat
     3001 ggcttctaca aagaccgcta cgtgtaccgc cacttcgcca gcgctgcact acccatcccc
     3061 gaagtgttgg acatcggcga gttcagcgag agcctgacat actgcatcag tagacgcgcc
     3121 caaggcgtta ctctccaaga cctccccgaa acagagctgc ctgctgtgtt acagcctgtc
     3181 gccgaagcta tggatgctat tgccgccgcc gacctcagtc aaaccagcgg cttcggccca
     3241 ttcgggcccc aaggcatcgg ccagtacaca acctggcggg atttcatttg cgccattgct
     3301 gatccccatg tctaccactg gcagaccgtg atggacgaca ccgtgtccgc cagcgtagct
     3361 caagccctgg acgaactgat gctgtgggcc gaagactgtc ccgaggtgcg ccacctcgtc
     3421 catgccgact tcggcagcaa caacgtcctg accgacaacg gccgcatcac cgccgtaatc
     3481 gactggtccg aagctatgtt cggggacagt cagtacgagg tggccaacat cttcttctgg
     3541 cggccctggc tggcttgcat ggagcagcag actcgctact tcgagcgccg gcatcccgag
     3601 ctggccggca gccctcgtct gcgagcctac atgctgcgca tcggcctgga tcagctctac
     3661 cagagcctcg tggacggcaa cttcgacgat gctgcctggg ctcaaggccg ctgcgatgcc
     3721 atcgtccgca gcggggccgg caccgtcggt cgcacacaaa tcgctcgccg gagcgcagcc
     3781 gtatggaccg acggctgcgt cgaggtgctg gccgacagcg gcaaccgccg gcccagtaca
     3841 cgaccgcgcg ctaaggaggt aggtcgagtt tagactctag ccatcacgat ggccgcaata
     3901 aaatatcttt attttcatta catctgtgtg ttggtttttt gtgtgaatcg atagcgataa
     3961 ggatcctctt tgcgcttgcg ttttcccttg tccagatagc ccagtagctg acattcatcc
     4021 ggggtcagca ccgtttctgc ggactggctt tctacgtaat ggtttcttag acgtcaggtg
     4081 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     4141 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     4201 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     4261 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     4321 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     4381 gccccgaaga acgttttcca atgatgagca ctttcaaagt tctgctatgt ggcgcggtat
     4441 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     4501 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     4561 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     4621 ctatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     4681 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     4741 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     4801 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     4861 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     4921 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     4981 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     5041 gtgcctcact gattaagcat tggtaattcg aaatgaccga ccaagcgacg cccaaccggt
     5101 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     5161 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     5221 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     5281 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     5341 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     5401 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     5461 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     5521 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     5581 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     5641 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     5701 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5761 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatttc aagaagatcc
     5821 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     5881 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttatagtccg gaaatacagg
     5941 aacgcacgct ggatggccct tcgctgggat ggtgaaacca tgaaaaatgg cagcttcagt
     6001 ggattaagtg ggggtaatgt ggcctgtacc ctctggttgc ataggtattc atacggttaa
     6061 aatttatcag gcgcgattgc ggcagttttt cgggtggttt gttgccattt ttacctgtct
     6121 gctgccgtga tcgcgctgaa cgcgttttag cggtgcgtac aattaaggga ttatggtaaa
     6181 tccacttact gtctgccctc gtagccatcg agataaaccg cagtactccg gccacgatgc
     6241 gtccggcgta gaggatcgag atct
//
