LOCUS       sequence-424500-        7227 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Insect expression of human RFWD3 fragment S425 to E774. N-terminal 
            biotin acceptor peptide tag and C-terminal 6X His tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7227)
  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 7227)
  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: pFBD-BirA_RFWD3:425-774
FEATURES             Location/Qualifiers
     source          1..7227
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      2..456
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(89..108)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     298..319
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        482..586
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(587..655,656..1447)
                     /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(805..824)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1618..2206
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2107..2126
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2360..2377
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  2511..2735
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(2802..3335)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(3505..3510,3511..3527,3528..3533))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     polyA_signal    4041..4089
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     4114..4133
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(4160..5125)
                     /codon_start=1
                     /gene="birA"
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             complement(4163..5125)
                     /codon_start=1
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     promoter        complement(5162..5271)
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     promoter        5295..5386
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     5311..5328
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     CDS             5437..5481
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             6565..6582
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    6717..6851
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6754..6773)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6808..6827
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  6880..7045
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
ORIGIN
        1 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
       61 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
      121 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
      181 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
      241 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt cttaatagtg
      301 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
      361 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
      421 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
      481 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
      541 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
      601 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
      661 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
      721 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
      781 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
      841 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
      901 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
      961 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     1021 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     1081 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     1141 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     1201 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     1261 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     1321 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     1381 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     1441 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     1501 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     1561 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     1621 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     1681 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     1741 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     1801 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     1861 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     1921 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     1981 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     2041 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     2101 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     2161 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     2221 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     2281 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     2341 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     2401 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atagaccagc cgcgtaacct
     2461 ggcaaaatcg gttacggttg agtaataaat ggatgccctg cgtaagcggg tgtgggcgga
     2521 caataaagtc ttaaactgaa caaaatagat ctaaactatg acaataaagt cttaaactag
     2581 acagaatagt tgtaaactga aatcagtcca gttatgctgt gaaaaagcat actggacttt
     2641 tgttatggct aaagcaaact cttcattttc tgaagtgcaa attgcccgtc gtattaaaga
     2701 ggggcgtggc caagggcatg gtaaagacta tattcgcggc gttgtgacaa tttaccgaac
     2761 aactccgcgg ccgggaagcc gatctcggct tgaacgaatt gttaggtggc ggtacttggg
     2821 tcgatatcaa agtgcatcac ttcttcccgt atgcccaact ttgtatagag agccactgcg
     2881 ggatcgtcac cgtaatctgc ttgcacgtag atcacataag caccaagcgc gttggcctca
     2941 tgcttgagga gattgatgag cgcggtggca atgccctgcc tccggtgctc gccggagact
     3001 gcgagatcat agatatagat ctcactacgc ggctgctcaa acttgggcag aacgtaagcc
     3061 gcgagagcgc caacaaccgc ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta
     3121 cggagcaagt tcccgaggta atcggagtcc ggctgatgtt gggagtaggt ggctacgtct
     3181 ccgaactcac gaccgaaaag atcaagagca gcccgcatgg atttgacttg gtcagggccg
     3241 agcctacatg tgcgaatgat gcccatactt gagccaccta actttgtttt agggcgactg
     3301 ccctgctgcg taacatcgtt gctgctccat aacatcaaac atcgacccac ggcgtaacgc
     3361 gcttgctgct tggatgcccg aggcatagac tgtacaaaaa aacagtcata acaagccatg
     3421 aaaaccgcca ctgcgccgtt accaccgctg cgttcggtca aggttctgga ccagttgcgt
     3481 gagcgcatac gctacttgca ttacagttta cgaaccgaac aggcttatgt caactgggtt
     3541 cgtgccttca tccgtttcca cggtgtgcgt cacccggcaa ccttgggcag cagcgaagtc
     3601 gaggcatttc tgtcctggct ggcgaacgag cgcaaggttt cggtctccac gcatcgtcag
     3661 gcattggcgg ccttgctgtt cttctacggc aaggtgctgt gcacggatct gcccttgctt
     3721 caggagatcg gtagacctcg gccgtcgcgg cgcttgccgg tggtgctgac cccggatgaa
     3781 gtggttcgca tcctcggttt tctggaaggc gagcatcgtt tgttcgccca ggactctagc
     3841 tatagttcta gtggttggct acgtacccgt agtggctatg gcagggcttg ccgccccgac
     3901 gttggctgcg agccctgggc cttcacccga acttgggggt tggggtgggg aaaaggaaga
     3961 aacgcgggcg tattggtccc aatggggtct cggtggggta tcgacagagt gccagccctg
     4021 ggaccgaacc ccgcgtttat gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt
     4081 ttattgccgt catagcgcgg gttccttccg gtattgtctc cttccgtgtt tcagttagcc
     4141 tcccccatct cccggtactt catttttctg cactacgcag ggatatttca ccgcccatcc
     4201 agggttttat tattccatcc tgctcaagta ataaagcccc ctgtttgtct attccgcgtg
     4261 aaatgccaaa tatttcttta tcaccaatga taagtttcac tgggcgatta ataaaattat
     4321 ccagcttttc ccagcgcgac agataaggtg ccaatccttc ttgttcgaag agttccaacg
     4381 cagcacgtaa ttcacgtatt agcatggccg ccaacgtatt acgatcgaga ttgatccccg
     4441 cttcctgcag cgtgatccac ccctgattaa cgacactctc ttcaacacgg cgcattgcca
     4501 tgttgatccc ggctccaatg actatttgcg ccgcatcgcc agttttgcca gtcagctcca
     4561 ccagaatgcc tgccagcttg cgatcctgca gatagaggtc attaggccat ttaacacgaa
     4621 ctttatctgc acccagcttg cgtaatactt ccgccatcac gataccgata accagactta
     4681 aaccaatcgc cgccgccggg ccttgttcca gacgccagaa catcgacaaa tataagtttg
     4741 cgccaaaagg cgaaaaccat ttccgacccc ggcgaccacg gccagcctgc tggtattctg
     4801 caatgcaagc atcgcccgat ttaagctctc cgatacgatc aagaaggtac tgattcgtgg
     4861 agtcaatcac tggcagcacg gctacactac cgccatccag ctgacccaat atctgtttag
     4921 cattaagtaa ctggataggc tcaggcaggc tgtatccttt acccggaacg gtaaagacat
     4981 caacgcccca gtcacgcagt gtctgaatgt gtttattaat agccgcccgg ctcattccca
     5041 gcgtttcacc caactgctcg ccagagtgaa attcaccgtt cgctaacagg gcaatcaatt
     5101 tcagtggcac ggtgttatcc ttcatggctc gagatcccgg gtgatcaagt cttcgtcgag
     5161 tgattgtaaa taaaatgtaa tttacagtat agtattttaa ttaatataca aatgatttga
     5221 taataattct tatttaacta taatatattg tgttgggttg aattaaaggt ccgtatactc
     5281 cggaatatta atagatcatg gagataatta aaatgataac catctcgcaa ataaataagt
     5341 attttactgt tttcgtaaca gttttgtaat aaaaaaacct ataaatattc cggattattc
     5401 ataccgtccc accatcgggc gcggatcccg atgagcggcc tgaacgatat ttttgaagcg
     5461 cagaaaattg aatggcatga aggcagcgct ggaggttcag gttcaccctc cagccagggc
     5521 cagcacaagc acaagtacca cttccaaaag accttcacag tatctcaggc aggaaactgc
     5581 cggatcatgg catactgtga tgctctgagc tgcctggtga tatcacagcc ttctcctcag
     5641 gcctcttttc ttccaggctt tggtgttaag atgttgagta ctgccaacat gaagagcagt
     5701 cagtacattc cgatgcatgg caaacagatc cgtggactgg cgtttagcag ttacctcaga
     5761 ggcttgctac tctctgcttc cctagacaac actattaaac tgaccagcct ggagacaaat
     5821 accgtggtcc agacttataa tgctggacgt cctgtctgga gctgttgctg gtgtcttgat
     5881 gaggctaact acatctatgc tggactggcc aatggttcaa ttctggtata tgacgtgcga
     5941 aacacgagca gtcatgtgca ggagttagta gctcagaaag ccagatgccc actggtctcc
     6001 ctgtcataca tgcccagagc tgcctcagct gcatttccat atggtggggt gctggctgga
     6061 accttggagg atgcttcatt ctgggaacag aaaatggact tttctcattg gcctcatgtg
     6121 ctgcccttgg agccaggggg ctgcatagac tttcagacag agaacagctc ccggcactgt
     6181 cttgtgacct acaggcctga taaaaatcac accaccatac gaagtgtgct gatggaaatg
     6241 tcctaccgac tggatgacac tggaaatcca atctgctcct gccagcctgt acatacattt
     6301 tttggaggac ctacttgcaa actattgacc aaaaatgcca ttttccaaag cccagagaat
     6361 gatggcaaca tcctggtgtg tactggggat gaagcagcaa attctgccct gctgtgggat
     6421 gctgccagtg gctcgttgct ccaggaccta cagaccgatc agcctgtgtt ggacatctgc
     6481 ccatttgagg tgaaccgtaa cagctacttg gctaccttaa cagagaagat ggtccacatc
     6541 tataagtggg agggcggaag tggacatcac caccatcatc actgatgaag cttgtcgaga
     6601 agtactagag gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa
     6661 acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact
     6721 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     6781 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     6841 atgtctggat ctgatcactg cttgagccta ggagatccga accagataag tgaaatctag
     6901 ttccaaacta ttttgtcatt tttaattttc gtattagctt acgacgctac acccagttcc
     6961 catctatttt gtcactcttc cctaaataat ccttaaaaac tccatttcca cccctcccag
     7021 ttcccaacta ttttgtccgc ccacagcggg gcatttttct tcctgttatg tttttaatca
     7081 aacatcctgc caactccatg tgacaaaccg tcatcttcgg ctactttttc tctgtcacag
     7141 aatgaaaatt tttctgtcat ctcttcgtta ttaatgtttg taattgactg aatatcaacg
     7201 cttatttgca gcctgaatgg cgaatgg
//
