LOCUS       sequence-473149-        7488 bp    DNA     circular SYN 19-MAY-2025
DEFINITION  transient overexpression of KBP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7488)
  AUTHORS   Donato V, Bonora M, Simoneschi D, Sartini D, Kudo Y, Saraf A, 
            Florens L, Washburn MP, Stadtfeld M, Pinton P, Pagano M
  TITLE     The TDH-GCN5L1-Fbxo15-KBP axis limits mitochondrial biogenesis in 
            mouse embryonic stem cells.
  JOURNAL   Nat Cell Biol. 2017 Apr;19(4):341-351. doi: 10.1038/ncb3491. Epub 
            2017 Mar 20.
  PUBMED    28319092
REFERENCE   2  (bases 1 to 7488)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 19, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1038/ncb3491"; 
            journalName: "Nat Cell Biol"; date: "2017-04"; volume: "19"; issue: 
            "4"; pages: "341-351"
COMMENT     Sequence Label: pcDNA3 Strep-FLAG KBP
FEATURES             Location/Qualifiers
     source          1..7488
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(125..919)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(236..255)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     846..865
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(986..1315)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1000..1135
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1300..1320
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(1329..1757)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1439..1460)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1651..1670
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    1803..2027
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     2016..2033
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     promoter        2053..2071
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2053..2070
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(2102..2125)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(2141..2164)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             complement(2201..2224)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     protein_bind    2238..2262
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(4126..4150)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(4230..4249)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4231..4249)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4294..4497)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4323..4343)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4498..4877
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5049..5068
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5143..5247
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5248..5316,5317..6108)
                     /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(5466..5485)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6279..6867
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6768..6787
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7021..7038
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7155..7176
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7191..7196,7197..7214,7215..7221)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7229..7245
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     7234..7256
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7253..7269
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7253..7269
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    7318..7439
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7329..7348)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     7383..7402
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
ORIGIN
        1 cagccggcgt cccggaaaac gattccgaag cccaaccttt catagaaggc ggcggtggaa
       61 tcgaaatctc gtgatggcag gttgggcgtc gcttggtcgg tcatttcgaa ccccagagtc
      121 ccgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa tcgggagcgg
      181 cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct tcagcaatat
      241 cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg ccacagtcga
      301 tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca tcgccatggg
      361 tcacgacgag atcctcgccg tcgggcatgc gcgccttgag cctggcgaac agttcggctg
      421 gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg gcttccatcc
      481 gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag gtagccggat
      541 caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg gcaggagcaa
      601 ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag tcccttcccg
      661 cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc agccacgata
      721 gccgcgctgc ctcgtcctgc agttcattca gggcaccgga caggtcggtc ttgacaaaaa
      781 gaaccgggcg cccctgcgct gacagccgga acacggcggc atcagagcag ccgattgtct
      841 gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa cctgcgtgca
      901 atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatca gatccgaaaa
      961 tggatataca agctcccggg agctttttgc aaaagcctag gcctccaaaa aagcctcctc
     1021 actacttctg gaatagctca gaggcagagg cggcctcggc ctctgcataa ataaaaaaaa
     1081 ttagtcagcc atggggcgga gaatgggcgg aactgggcgg agttaggggc gggatgggcg
     1141 gagttagggg cgggactatg gttgctgact aattgagatg catgctttgc atacttctgc
     1201 ctgctgggga gcctggggac tttccacacc tggttgctga ctaattgaga tgcatgcttt
     1261 gcatacttct gcctgctggg gagcctgggg actttccaca ccctaactga cacacattcc
     1321 acagaattaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     1381 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     1441 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     1501 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     1561 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     1621 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     1681 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     1741 gccgctacag ggcgcgtggg gataccccct agagccccag ctggttcttt ccgcctcaga
     1801 agccatagag cccaccgcat ccccagcatg cctgctattg tcttcccaat cctccccctt
     1861 gctgtcctgc cccaccccac cccccagaat agaatgacac ctactcagac aatgcgatgc
     1921 aatttcctca ttttattagg aaaggacagt gggagtggca ccttccaggg tcaaggaagg
     1981 cacgggggag gggcaaacaa cagatggctg gcaactagaa ggcacagtcg aggctgatca
     2041 gcgagctcta gcatttaggt gacactatag aatagggccc tctagatgca tgctcgagtc
     2101 atttatcatc atcatcttta taatcctctc cgctagctcc tttctcgaac tgcgggtggc
     2161 tccagcttcc tcctccgctg cctccgccgc ttcctcctcc cttctcgaac tgagggtggc
     2221 tccagctggc ggatcgaacc actttgtaca agaaagttgg gtaagtcagg gccatcttgg
     2281 ttctgaatct ctccattttt gttgggagaa gactaaccat ctctttacta agttctagct
     2341 caacttctat ttcctgggcg gcctcaggat gcttttcaca gtaatcaaca ataaatttgt
     2401 aatgttccaa tgatgttgcc aaattttcca gctctttctt gggatctgca gtaatgattt
     2461 tgccatagag acgggcaact cgaaacttag ctaacatggc agggcgaaga acatcttccc
     2521 ctatatgctc agggaatact ttatttgggt ctctcaggga gtctaagaag agctggtagt
     2581 acttcagtgc tgacttatta agattattta ttttttttac aatgtgtgaa tcaggatccc
     2641 ttagcctgtc agcaatggca accttcaaat ccatcatatc atagtaagca tgtgcaattt
     2701 caaactggat ctgtctgttg accaacagat aatactgtgg attcaggtct acagttaggg
     2761 gctctagcat ggctattctg cgtttatgca tcttgcaccg tctctccatg tcagtttcaa
     2821 agaatgcaag caccttaaac agagcactgt ggtcttggac aacttcaata tggtcagtga
     2881 cataaccatc aatctgaaag aactcttttg cctcaaagac atagtgctga cccaataaga
     2941 aaagttctct ggcttcttca aaatctaaag gtctcaagta gctcactttc tcttctactg
     3001 cagagatggc atcacacagt tcaccggttc caaactgcac agcttttttc cgaatgcttt
     3061 cctcctcatc tagttctttt ttccttaaag ctctaagttc agactgttta tcaagatcaa
     3121 gctctcctat gttgtcctgc atggagagtt gggcattctg catgagagtc aaacagtatt
     3181 tgatccagca ccttgctatt tccccctttc tttgatgata aagctctggc acttctcctt
     3241 cagcttcagg agtgtcttct gtggctgaga tctttccagt ttgaccaaaa atgacattag
     3301 cagctgataa acagtgcctg gcctccataa agcatagctt attgatgtaa aactgtgaca
     3361 aggtagcagc attgatagcc cactctatag gatggtaggc attgtgctca agctggcgtt
     3421 ttagtgtact atggcaatag tgagcagcct tctcaaacat ttccagatgc tggtagactt
     3481 gagctaggta atataggtta tgagtataaa ccttttcaaa tctttttgat ctctcttgtt
     3541 cagtaagttt ctcttcttca ggaagaaaac gctcagtagg atcaagagga ggactcccaa
     3601 cctctttcat atactgatta tatagtgctt ctgatgactc taggtaagcc tgtgcagttt
     3661 caatttcttc tctttcagac cacaagatac ccaggttatt ctgcgcctgg atgcagagag
     3721 agatgcagtc gtgcgagagc cggtacctgc gcagcagccg caggcatttc accaggtgct
     3781 cctcccccgc cgacagctcc tccgtgtcga tgtggttcac cccgaggtgg aactcgatga
     3841 ctgccagcct caccgctcgc tgggcgacgg gcccctcggg ctccaccacc tcagccggca
     3901 gccccagggc gtggtcaccg gcacccgggc cgtcctcggc ctcaggccgc tcatcctcgt
     3961 cctcaggcgc agggccgagc agcgccttga cctcttccag tagcgcccgg gcgctgtatt
     4021 tggacttgta tggttccttc tccggatttt tatgcagttc cacccgcgac agagcgagcg
     4081 ccgcctggaa tttctcgcag acctccgccc acggaacgtt cgccatgcca acttttttgt
     4141 acaaacttgt ttgatccgcg gtggcggcga attccagcac actggcggcc gttactagtg
     4201 gatccgagct cggtaccaag cttgggtctc cctatagtga gtcgtattaa tttcgataag
     4261 ccagtaagca gtgggttctc tagttagcca gagagctctg cttatataga cctcccaccg
     4321 tacacgccta ccgcccattt gcgtcaatgg ggcggagttg ttacgacatt ttggaaagtc
     4381 ccgttgattt tggtgccaaa acaaactccc attgacgtca atggggtgga gacttggaaa
     4441 tccccgtgag tcaaaccgct atccacgccc attgatgtac tgccaaaacc gcatcaccat
     4501 ggtaatagcg atgactaata cgtagatgta ctgccaagta ggaaagtccc ataaggtcat
     4561 gtactgggca taatgccagg cgggccattt accgtcattg acgtcaatag ggggcgtact
     4621 tggcatatga tacacttgat gtactgccaa gtgggcagtt taccgtaaat actccaccca
     4681 ttgacgtcaa tggaaagtcc ctattggcgt tactatggga acatacgtca ttattgacgt
     4741 caatgggcgg gggtcgttgg gcggtcagcc aggcgggcca tttaccgtaa gttatgtaac
     4801 gcggaactcc atatatgggc tatgaactaa tgaccccgta attgattact attaataact
     4861 agtcaataat caatgtcaac gcgtatatct ggcccgtaca tcgcgaagca gcgcaaaacg
     4921 cctaacccta agcagattct tcatgcaatt gtcggtcaag ccttgccttg ttgtagctta
     4981 aattttgctc gcgcactact cagcgacctc caacacacaa gcagggagca gatactggct
     5041 taactatgcg gcatcagagc agattgtact gagagtgcac cataggggat cgggagatct
     5101 cccgatccgt cgacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     5161 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     5221 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     5281 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     5341 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     5401 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     5461 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     5521 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     5581 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     5641 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     5701 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     5761 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     5821 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     5881 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     5941 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     6001 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     6061 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     6121 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     6181 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     6241 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     6301 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     6361 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     6421 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     6481 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     6541 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6601 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6661 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6721 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6781 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     6841 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     6901 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     6961 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     7021 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     7081 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     7141 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
     7201 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
     7261 agctatgacc atgattacgc caagctctag ctagaggtcg acggtataca gacatgataa
     7321 gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt
     7381 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagttg
     7441 gggtgggcga agaactccag catgagatcc ccgcgctgga ggatcatc
//
