LOCUS       sequence-425982-        7593 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of full-length human AIRE. C-terminal NanoLuc 
            luciferase tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7593)
  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 7593)
  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_AIRE:1-545
FEATURES             Location/Qualifiers
     source          1..7593
                     /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             2768..3280
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     primer_bind     complement(3356..3374)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    3411..3532
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3422..3441)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3476..3495
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3777..4134
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3777..3968
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3800..3820)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3985..4120
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4165..5202
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             5181..5189
                     /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    5226..5274
                     /note="synthetic polyadenylation signal"
     promoter        5430..5534
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5535..5603,5604..6395)
                     /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(5753..5772)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(6553..7141)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6633..6652)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    7257..7540
                     /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 caccatggcg acggacgcgg cgctacgccg gcttctgagg ctgcaccgca cggagatcgc
     1141 ggtggccgtg gacagcgcct tcccactgct gcacgcgctg gctgaccacg acgtggtccc
     1201 cgaggacaag tttcaggaga cgcttcatct gaaggaaaag gagggctgcc cccaggcctt
     1261 ccacgccctc ctgtcctggc tgctgaccca ggactccaca gccatcctgg acttctggag
     1321 ggtgctgttc aaggactaca acctggagcg ctatggccgg ctgcagccca tcctggacag
     1381 cttccccaaa gatgtggacc tcagccagcc ccggaagggg aggaagcccc cggccgtccc
     1441 caaggctttg gtaccgccac ccagactccc caccaagagg aaggcctcag aagaggctcg
     1501 agctgccgcg ccagcagccc tgactccaag gggcaccgcc agcccaggct ctcaactgaa
     1561 ggccaagccc cccaagaagc cggagagcag cgcagagcag cagcgccttc cactcgggaa
     1621 cgggattcag accatgtcag cttcagtcca gagagctgtg gccatgtcct ccggggacgt
     1681 cccgggagcc cgaggggccg tggaggggat cctcatccag caggtgtttg agtcaggcgg
     1741 ctccaagaag tgcatccagg ttggtgggga gttctacact cccagcaagt tcgaagactc
     1801 cggcagtggg aagaacaagg cccgcagcag cagtggcccg aagcctctgg ttcgagccaa
     1861 gggagcccag ggcgctgccc ccggtggagg tgaggctagg ctgggccagc agggcagcgt
     1921 tcccgcccct ctggccctcc ccagtgaccc ccagctccac cagaagaatg aggacgagtg
     1981 tgccgtgtgt cgggacggcg gggagctcat ctgctgtgac ggctgccctc gggccttcca
     2041 cctggcctgc ctgtcccctc cgctccggga gatccccagt gggacctgga ggtgctccag
     2101 ctgcctgcag gcaacagtcc aggaggtgca gccccgggca gaggagcccc ggccccagga
     2161 gccacccgtg gagaccccgc tccccccggg gcttaggtcg gcgggagagg aggtaagagg
     2221 tccacctggg gaacccctag ccggcatgga cacgactctt gtctacaagc acctgccggc
     2281 tccgccttct gcagccccgc tgccagggct ggactcctcg gccctgcacc ccctactgtg
     2341 tgtgggtcct gagggtcagc agaacctggc tcctggtgcg cgttgcgggg tgtgcggaga
     2401 tggtacggac gtgctgcggt gtactcactg cgccgctgcc ttccactggc gctgccactt
     2461 cccagccggc acctcccggc ccgggacggg cctgcgctgc agatcctgct caggagacgt
     2521 gaccccagcc cctgtggagg gggtgctggc ccccagcccc gcccgcctgg cccctgggcc
     2581 tgccaaggat gacactgcca gtcacgagcc cgctctgcac agggatgacc tggagtccct
     2641 tctgagcgag cacaccttcg atggcatcct gcagtgggcc atccagagca tggcccgtcc
     2701 ggcggccccc ttcccctcct ctagatttgg gcccaattcc tgcaggcgag ctctcggctc
     2761 gagcggcgtc ttcacactcg aagatttcgt tggggactgg cgacagacag ccggctacaa
     2821 cctggaccaa gtccttgaac agggaggtgt gtccagtttg tttcagaatc tcggggtgtc
     2881 cgtaactccg atccaaagga ttgtcctgag cggtgaaaat gggctgaaga tcgacatcca
     2941 tgtcatcatc ccgtatgaag gtctgagcgg cgaccaaatg ggccagatcg aaaaaatttt
     3001 taaggtggtg taccctgtgg atgatcatca ctttaaggtg atcctgcact atggcacact
     3061 ggtaatcgac ggggttacgc cgaacatgat cgactatttc ggacggccgt atgaaggcat
     3121 cgccgtgttc gacggcaaaa agatcactgt aacagggacc ctgtggaacg gcaacaaaat
     3181 tatcgacgag cgcctgatca accccgacgg ctccctgctg ttccgagtaa ccatcaacgg
     3241 agtgaccggc tggcggctgt gcgaacgcat tctggcgtaa ggccgcgact ctagggcatg
     3301 caagctgatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     3361 gagcaataac tagcataacc ccttggggcg gccgcttcga gcagacatga taagatacat
     3421 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
     3481 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa
     3541 caattgcatt cattttatgt ttcaggttca gggggagatg tgggaggttt ttttaagcaa
     3601 gtaaaacctc tacaaatgtg gtaaaatcga attttaacaa aatattaacg cttacaattt
     3661 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgcggatc
     3721 tgcgcagcac catggcctga aataacctct gaaagaggaa cttggttagg taccttctga
     3781 ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     3841 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     3901 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3961 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     4021 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     4081 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     4141 gattcttctg acactagcgc caccatgaag aagcccgaac tcaccgctac cagcgttgaa
     4201 aaatttctca tcgagaagtt cgacagtgtg agcgacctga tgcagttgtc ggagggcgaa
     4261 gagagccgag ccttcagctt cgatgtcggc ggacgcggct atgtactgcg ggtgaatagc
     4321 tgcgctgatg gcttctacaa agaccgctac gtgtaccgcc acttcgccag cgctgcacta
     4381 cccatccccg aagtgttgga catcggcgag ttcagcgaga gcctgacata ctgcatcagt
     4441 agacgcgccc aaggcgttac tctccaagac ctccccgaaa cagagctgcc tgctgtgtta
     4501 cagcctgtcg ccgaagctat ggatgctatt gccgccgccg acctcagtca aaccagcggc
     4561 ttcggcccat tcgggcccca aggcatcggc cagtacacaa cctggcggga tttcatttgc
     4621 gccattgctg atccccatgt ctaccactgg cagaccgtga tggacgacac cgtgtccgcc
     4681 agcgtagctc aagccctgga cgaactgatg ctgtgggccg aagactgtcc cgaggtgcgc
     4741 cacctcgtcc atgccgactt cggcagcaac aacgtcctga ccgacaacgg ccgcatcacc
     4801 gccgtaatcg actggtccga agctatgttc ggggacagtc agtacgaggt ggccaacatc
     4861 ttcttctggc ggccctggct ggcttgcatg gagcagcaga ctcgctactt cgagcgccgg
     4921 catcccgagc tggccggcag ccctcgtctg cgagcctaca tgctgcgcat cggcctggat
     4981 cagctctacc agagcctcgt ggacggcaac ttcgacgatg ctgcctgggc tcaaggccgc
     5041 tgcgatgcca tcgtccgcag cggggccggc accgtcggtc gcacacaaat cgctcgccgg
     5101 agcgcagccg tatggaccga cggctgcgtc gaggtgctgg ccgacagcgg caaccgccgg
     5161 cccagtacac gaccgcgcgc taaggaggta ggtcgagttt agactctagc catcacgatg
     5221 gccgcaataa aatatcttta ttttcattac atctgtgtgt tggttttttg tgtgaatcga
     5281 tagcgataag gatcctcttt gcgcttgcgt tttcccttgt ccagatagcc cagtagctga
     5341 cattcatccg gggtcagcac cgtttctgcg gactggcttt ctacgtaatg gtttcttaga
     5401 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     5461 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     5521 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     5581 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     5641 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5701 agagttttcg ccccgaagaa cgttttccaa tgatgagcac tttcaaagtt ctgctatgtg
     5761 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5821 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     5881 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5941 ttctgacaac tatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     6001 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     6061 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     6121 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     6181 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     6241 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     6301 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     6361 ctgagatagg tgcctcactg attaagcatt ggtaattcga aatgaccgac caagcgacgc
     6421 ccaaccggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     6481 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     6541 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     6601 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     6661 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     6721 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     6781 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     6841 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     6901 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     6961 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct
     7021 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     7081 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatttca
     7141 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     7201 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt tatagtccgg
     7261 aaatacagga acgcacgctg gatggccctt cgctgggatg gtgaaaccat gaaaaatggc
     7321 agcttcagtg gattaagtgg gggtaatgtg gcctgtaccc tctggttgca taggtattca
     7381 tacggttaaa atttatcagg cgcgattgcg gcagtttttc gggtggtttg ttgccatttt
     7441 tacctgtctg ctgccgtgat cgcgctgaac gcgttttagc ggtgcgtaca attaagggat
     7501 tatggtaaat ccacttactg tctgccctcg tagccatcga gataaaccgc agtactccgg
     7561 ccacgatgcg tccggcgtag aggatcgaga tct
//
