LOCUS       sequence-245656-        7518 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of Skp2 WT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7518)
  AUTHORS   Shin HR, Kim H, Oh S, Lee JG, Kee M, Ko HJ, Kweon MN, Won KJ, Baek 
            SH
  TITLE     AMPK-SKP2-CARM1 signalling cascade in transcriptional regulation of 
            autophagy.
  JOURNAL   Nature. 2016 Jun 15. doi: 10.1038/nature18014.
  PUBMED    27309807
REFERENCE   2  (bases 1 to 7518)
  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: "Nature. 
            2016 Jun 15. doi: 10.1038/nature18014."
COMMENT     Sequence Label: CMV10-3xFlag Skp2 WT
FEATURES             Location/Qualifiers
     source          1..7518
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             614..679
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    1960..2582
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2159..2178)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(2606..2626)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2624..2921
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             2975..3769
                     /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(3029..3048)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3639..3658
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4421..4555
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4458..4477)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4512..4531
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4560..4579)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4561..4579)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4593..4609)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4593..4609)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4606..4628)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4617..4633
                     /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(4641..4647,4648..4665,4666..4671))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4686..4707
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4824..4841)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4995..5583)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5075..5094)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5754..6545,6546..6614))
                     /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     6377..6396
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6615..6719)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6746..7201
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6833..6852)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7043..7064
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7327..7349
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7341..7358
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7342..7358
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt ccgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta caccaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaataa ccccgccccg ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc
      601 agaattaacc atggactaca aagaccatga cggtgattat aaagatcatg acatcgatta
      661 caaggatgac gatgacaagc ttgcggccgc gaattcacac aggaagcacc tccaggagat
      721 tccagacctg agtagcaacg ttgccaccag cttcacgtgg ggatgggatt ccagcaagac
      781 ttctgaactg ctgtcaggca tgggggtctc cgccctggag aaagaggagc ccgacagtga
      841 gaacatcccc caggaactgc tctcaaacct gggccacccg gagagccccc cacggaaacg
      901 gctgaagagc aaagggagtg acaaagactt tgtgattgtc cgcaggccta agctaaatcg
      961 agagaacttt ccaggtgttt catgggactc ccttccggat gagctgctct tgggaatctt
     1021 ttcctgtctg tgcctccctg agctgctaaa ggtctctggt gtttgtaaga ggtggtatcg
     1081 cctagcgtct gatgagtctc tatggcagac cttagacctc acaggtaaaa atctgcaccc
     1141 ggatgtgact ggtcggttgc tgtctcaagg ggtgattgcc ttccgctgcc cacgatcatt
     1201 tatggaccaa ccattggctg aacatttcag cccttttcgt gtacagcaca tggacctatc
     1261 gaactcagtt atagaagtgt ccaccctcca cggcatactg tctcagtgtt ccaagttgca
     1321 gaatctaagc ctggaaggcc tgcggctttc ggatcccatt gtcaatactc tcgcaaaaaa
     1381 ctcaaattta gtgcgactta acctttctgg gtgttctgga ttctctgaat ttgccctgca
     1441 gactttgcta agcagctgtt ccagactgga tgagctgaac ctctcctggt gttttgattt
     1501 cactgaaaag catgtacagg tggctgttgc gcatgtgtca gagaccatca cccagctgaa
     1561 tcttagcggc tacagaaaga atctccagaa atcagatctc tctactttag ttagaagatg
     1621 ccccaatctt gtccatctag acttaagtga tagtgtcatg ctaaagaatg actgctttca
     1681 ggaatttttc cagctcaact acctccaaca cctatcactc agtcggtgct atgatataat
     1741 acctgaaact ttactattag tgacaagagc tggggttagg atccggttgg actctgacat
     1801 cggatgccct caaacataca gaacttccaa actcaagtcc agccataagc tattttgcca
     1861 acatgtcaga gtaatctgta tttttgtatg tgatttctac ttttatagac ttgttttaaa
     1921 acaataagat atcggtacca gtcgactcta gaggatcccg ggtggcatcc ctgtgacccc
     1981 tccccagtgc ctctcctggc cctggaagtt gccactccag tgcccaccag ccttgtccta
     2041 ataaaattaa gttgcatcat tttgtctgac taggtgtcct tctataatat tatggggtgg
     2101 aggggggtgg tatggagcaa ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc
     2161 tattgggaac caagctggag tgcagtggca caatcttggc tcactgcaat ctccgcctcc
     2221 tgggttcaag cgattctcct gcctcagcct cccgagttgt tgggattcca ggcatgcatg
     2281 accaggctca gctaattttt gtttttttgg tagagacggg gtttcaccat attggccagg
     2341 ctggtctcca actcctaatc tcaggtgatc tacccacctt ggcctcccaa attgctggga
     2401 ttacaggcgt gaaccactgc tcccttccct gtccttctga ttttaaaata actataccag
     2461 caggaggacg tccagacaca gcataggcta cctggccatg cccaaccggt gggacatttg
     2521 agttgcttgc ttggcactgt cctctcatgc gttgggtcca ctcagtagat gcctgttgaa
     2581 ttgggtacgc ggccagcttg gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     2641 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     2701 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     2761 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     2821 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     2881 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggaattga tcagcttggg
     2941 atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     3001 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     3061 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     3121 caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg
     3181 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     3241 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     3301 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     3361 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     3421 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     3481 actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg
     3541 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     3601 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     3661 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     3721 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     3781 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     3841 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     3901 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccgggctcga tcccctcgcg
     3961 agttggttca gctgctgcct gaggctggac gacctcgcgg agttctaccg gcagtgcaaa
     4021 tccgtcggca tccaggaaac cagcagcggc tatccgcgca tccatgcccc cgaactgcag
     4081 gagtggggag gcacgatggc cgctttggtc gacccggacg ggacgctcct gcgcctgata
     4141 cagaacgaat tgcttgcagg catctcatga gtgtgtcttc ccgttttccg cctgaggtca
     4201 ctgcgtggat ggagcgctgg cgcctgctgc gcgacggcga gctgctcacc acccactcgc
     4261 caagctggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgccgatcat
     4321 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc
     4381 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta
     4441 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     4501 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatcaattc
     4561 cctatagtga gtcgtattaa attcgtaatc atgtcatagc tgtttcctgt gtgaaattgt
     4621 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     4681 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     4741 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     4801 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4861 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4921 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4981 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5041 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5101 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5161 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     5221 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5281 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     5341 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     5401 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     5461 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     5521 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     5581 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     5641 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     5701 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     5761 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     5821 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     5881 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     5941 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     6001 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     6061 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     6121 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     6181 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     6241 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     6301 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     6361 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     6421 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     6481 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     6541 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     6601 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     6661 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     6721 acatttcccc gaaaagtgcc acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt
     6781 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     6841 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     6901 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     6961 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     7021 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     7081 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     7141 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     7201 tccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
     7261 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
     7321 ggttttccca gtcacgacgt tgtaaaacga cggccagtgc caagctgatc tatacattga
     7381 atcaatattg gcaattagcc atattagtca ttggttatat agcataaatc aatattggct
     7441 attggccatt gcatacgttg tatctatatc ataatatgta catttatatt ggctcatgtc
     7501 caatatgacc gccatgtt
//
