LOCUS       sequence-422389-        5765 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human SKP1 in bacteria cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5765)
  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 5765)
  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: pET-24d-Flag-SKP1
FEATURES             Location/Qualifiers
     source          1..5765
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(671..694)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     RBS             708..730
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    745..769
                     /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     complement(769..788)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(770..788)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(872..891)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1097..1174
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     complement(1194..1213)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3064..3255
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    3357..3496
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3412..3431
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3511..3528)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3682..4270)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3762..3781)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4381..4400)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4392..5207
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             4392..5207
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4465..4484)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5299..5754)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5436..5457)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5648..5667
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gctcacttct
      181 cttcacacca ctggttctct ttgcgtacct gggcttcctc ctcttcagta aagtcatttt
      241 tgatattgaa ggtcttgcga atctcctcag gagttttccc cttgatcata ttggcaacag
      301 tcttgcatgt aacatcaagc aaacctttga tgtctaagta gtttgcagcc agaatgagtt
      361 caaaaagtgt tccttggtca actttcagga attcttggtc ccaaacaggg atatcatctg
      421 ttcgcttttc tttgttctca tcatcttcag gaggaggagg gtcatccttg tggtgggtgc
      481 accactgaat gacctttttt aatattgctg cattcacatt tggtagagga actgggtcat
      541 catctccttc atcatccatt cccaaatctt ccaacatggt cttaatagtc acagattgtt
      601 tggcaatttc cacatcaact tcaaatatct ctccatcaga actctgcaac ttaattgaag
      661 gcattcctcc cttgtcatcg tcgtccttgt agtccatcca tggtatatct ccttcttaaa
      721 gttaaacaaa attatttcta gaggggaatt gttatccgct cacaattccc ctatagtgag
      781 tcgtattaat ttcgcgggat cgagatctcg atcctctacg ccggacgcat cgtggccggc
      841 atcaccggcg ccacaggtgc ggttgctggc gcctatatcg ccgacatcac cgatggggaa
      901 gatcgggctc gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc
      961 cccgtggccg ggggactgtt gggcgccatc tccttgcatg caccattcct tgcggcggcg
     1021 gtgctcaacg gcctcaacct actactgggc tgcttcctaa tgcaggagtc gcataaggga
     1081 gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata
     1141 gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt
     1201 cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca
     1261 cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc
     1321 caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc
     1381 cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca
     1441 actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc
     1501 ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga
     1561 tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga
     1621 tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact
     1681 gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt
     1741 aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatctcact cgcaatcaaa
     1801 ttcagccgat agcggaacgg gaaggcgact ggagtgccat gtccggtttt caacaaacca
     1861 tgcaaatgct gaatgagggc atcgttccca ctgcgatgct ggttgccaac gatcagatgg
     1921 cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg gatatctcgg
     1981 tagtgggata cgacgatacc gaagacagct catgttatat cccgccgtta accaccatca
     2041 aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg
     2101 gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc
     2161 tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
     2221 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtaagttag
     2281 ctcactcatt aggcaccggg atctcgaccg atgcccttga gagccttcaa cccagtcagc
     2341 tccttccggt gggcgcgggg catgactatc gtcgccgcac ttatgactgt cttctttatc
     2401 atgcaactcg taggacaggt gccggcagcg ctctgggtca ttttcggcga ggaccgcttt
     2461 cgctggagcg cgacgatgat cggcctgtcg cttgcggtat tcggaatctt gcacgccctc
     2521 gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg gcgagaagca ggccattatc
     2581 gccggcatgg cggccccacg ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta
     2641 ggctggcggg gttgccttac tggttagcag aatgaatcac cgatacgcga gcgaacgtga
     2701 agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt cttcggtttc
     2761 cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct gcaccattat gttccggatc
     2821 tgcatcgcag gatgctgctg gctaccctgt ggaacaccta catctgtatt aacgaagcgc
     2881 tggcattgac cctgagtgat ttttctctgg tcccgccgca tccataccgc cagttgttta
     2941 ccctcacaac gttccagtaa ccgggcatgt tcatcatcag taacccgtat cgtgagcatc
     3001 ctctctcgtt tcatcggtat cattaccccc atgaacagaa atccccctta cacggaggca
     3061 tcagtgacca aacaggaaaa aaccgccctt aacatggccc gctttatcag aagccagaca
     3121 ttaacgcttc tggagaaact caacgagctg gacgcggatg aacaggcaga catctgtgaa
     3181 tcgcttcacg accacgctga tgagctttac cgcagctgcc tcgcgcgttt cggtgatgac
     3241 ggtgaaaacc tctgacacat gcggctcccg gagacggtca cagcttgtct gtaagcggat
     3301 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca
     3361 gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat gcggcatcag
     3421 agcagattgt actgagagtg caccattgcg gtgtgaaata ccgcacagat gcgtaaggag
     3481 aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     3541 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     3601 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     3661 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     3721 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     3781 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     3841 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     3901 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     3961 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     4021 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     4081 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
     4141 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     4201 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     4261 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     4321 ctcacgttaa gggattttgg tcatgaacaa taaaactgtc tgcttacata aacagtaata
     4381 caaggggtgt tatgagccat attcaacggg aaacgtcttg ctctaggccg cgattaaatt
     4441 ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag
     4501 gtgcgacaat ctatcgattg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg
     4561 gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg
     4621 aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac
     4681 tcaccactgc gatccccggg aaaacagcat tccaggtatt agaagaatat cctgattcag
     4741 gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt
     4801 gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga
     4861 ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac
     4921 aagtctggaa agaaatgcat aaacttttgc cattctcacc ggattcagtc gtcactcatg
     4981 gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg
     5041 ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg
     5101 gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg
     5161 atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaagaa ttaattcatg
     5221 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     5281 ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     5341 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     5401 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     5461 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     5521 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     5581 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     5641 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     5701 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt
     5761 cgcca
//
