LOCUS       sequence-424286-        5903 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human SKP1 K142R/K163R mutant in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5903)
  AUTHORS   Li J, Krause GJ, Gui Q, Kaushik S, Rona G, Zhang Q, Liang FX, 
            Dhabaria A, Anerillas C, Martindale JL, Vasilyev N, Askenazi M, 
            Ueberheide B, Nudler E, Gorospe M, Cuervo AM, Pagano M
  TITLE     A noncanonical function of SKP1 regulates the switch between 
            autophagy and unconventional secretion.
  JOURNAL   Sci Adv. 2023 Oct 13;9(41):eadh1134. doi: 10.1126/sciadv.adh1134. 
            Epub 2023 Oct 13.
  PUBMED    37831778
REFERENCE   2  (bases 1 to 5903)
  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"; journalName: "Sci Adv."; 
            date: "2023-10-13"; volume: "9(41):eadh1134. doi"; pages: " 
            10.1126/sciadv.adh1134. Epub 2023 Oct 13"
COMMENT     Sequence Label: Flag-SKP1-RR (K142R/K163R)
FEATURES             Location/Qualifiers
     source          1..5903
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             916..939
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(1456..1474)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1457..1474)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1494..1511)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1500..1724
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1770..2198
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1857..1876)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2067..2088
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2207..2227)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2212..2541
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2392..2527
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2454..2473
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2608..3402
                     /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(2662..2681)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3272..3291
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3576..3697
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3613..3632)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3667..3686
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3746..3762)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3746..3762)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3759..3781)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3770..3786
                     /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)."
     promoter        complement(join(3794..3800,3801..3818,3819..3824))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3839..3860
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3977..3994)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4148..4736)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4228..4247)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4907..5698,5699..5767))
                     /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     5530..5549
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5768..5872)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcggat ccatggatta caaggatgac gacgataagc cttcaattaa gttgcagagt
      961 tctgatggag agatatttga agttgatgtg gaaattgcca aacaatctgt gactattaag
     1021 accatgttgg aagatttggg aatggatgat gaaggagatg atgacccagt tcctctacca
     1081 aatgtgaatg cagcaatatt aaaaaaggtc attcagtggt gcacccacca caaggatgac
     1141 cctcctcctc ctgaagatga tgagaacaaa gaaaagcgaa cagatgatat ccctgtttgg
     1201 gaccaagaat tcctgaaagt tgaccaagga acactttttg aactcattct ggctgcaaac
     1261 tacttagaca tcaaaggttt gcttgatgtt acatgcaaga ctgttgccaa tatgatcaag
     1321 gggaaaactc ctgaggagat tcgcaagacc ttcaatatca gaaatgactt tactgaagag
     1381 gaggaagccc aggtacgcaa agagaaccag tggtgtgaag agaggtgact cgagcatgca
     1441 tctagagggc cctattctat agtgtcacct aaatgctaga gctcgctgat cagcctcgac
     1501 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     1561 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     1621 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     1681 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     1741 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     1801 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     1861 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     1921 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     1981 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     2041 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     2101 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     2161 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     2221 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     2281 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     2341 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     2401 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     2461 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     2521 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     2581 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     2641 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     2701 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     2761 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     2821 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     2881 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     2941 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     3001 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     3061 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     3121 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     3181 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     3241 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     3301 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     3361 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     3421 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     3481 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     3541 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     3601 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     3661 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     3721 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     3781 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     3841 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     3901 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     3961 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     4021 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     4081 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     4141 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     4201 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     4261 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     4321 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     4381 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     4441 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     4501 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     4561 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     4621 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4681 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     4741 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4801 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     4861 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     4921 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     4981 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     5041 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     5101 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     5161 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     5221 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     5281 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     5341 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     5401 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     5461 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     5521 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     5581 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     5641 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     5701 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     5761 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     5821 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     5881 cccgaaaagt gccacctgac gtc
//
