LOCUS       sequence-450550-        5416 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-expression of FBXL5-SKP1 complex in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5416)
  AUTHORS   Wang H, Shi H, Rajan M, Canarie ER, Hong S, Simoneschi D, Pagano M, 
            Bush MF, Stoll S, Leibold EA, Zheng N
  TITLE     FBXL5 Regulates IRP2 Stability in Iron Homeostasis via an 
            Oxygen-Responsive [2Fe2S] Cluster.
  JOURNAL   Mol Cell. 2020 Apr 2;78(1):31-41.e5. doi: 
            10.1016/j.molcel.2020.02.011. Epub 2020 Mar 2.
  PUBMED    32126207
REFERENCE   2  (bases 1 to 5416)
  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: 
            "10.1016/j.molcel.2020.02.011"; journalName: "Mol Cell"; date: 
            "2020-04-2- 2"; volume: "78"; issue: "1"; pages: "31-41.e5"
COMMENT     Sequence Label: pRSFDuet-FBXL5-SKP1
FEATURES             Location/Qualifiers
     source          1..5416
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1..816)
                     /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             complement(1..816)
                     /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     724..743
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(817..908)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(924..1673)
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     complement(934..951)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(1174..1193)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1869..2951)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     2913..2932
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2952..3029)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        3075..3093
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3094..3118
                     /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)."
     CDS             3174..3191
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3201..3218
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3228..3389
                     /codon_start=1
                     /product="B1 domain of Streptococcal protein G   "
                     /label=GB1
                     /note="effective as a solubilizing fusion partner (Cheng 
                     and Patel, 2004)"
                     /translation="YKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDD
                     ATKTFTVTE"
     CDS             3393..3413
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     promoter        4448..4466
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4467..4491
                     /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)."
     CDS             5044..5088
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(5126..5144)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5140..5187
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
       61 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
      121 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
      181 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
      241 tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca
      301 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
      361 cgcctgagcg agacgaaata cgcggtcgct gttaaaagga caattacaaa caggaatcga
      421 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
      481 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
      541 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
      601 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
      661 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
      721 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
      781 cctagagcaa gacgtttccc gttgaatatg gctcatactc ttcctttttc aatattattg
      841 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
      901 taaacaaata ggcatgcagc gctcttccgc ttcctcgctc actgactcgc tacgctcggt
      961 cgttcgactg cggcgagcgg tgtcagctca ctcaaaagcg gtaatacggt tatccacaga
     1021 atcaggggat aaagccggaa agaacatgtg agcaaaaagc aaagcaccgg aagaagccaa
     1081 cgccgcaggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     1141 caagccagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     1201 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     1261 tcccttcggg aagcgtggcg ctttctcata gctcacgctg ttggtatctc agttcggtgt
     1321 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     1381 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     1441 cagcagccat tggtaactga tttagaggac tttgtcttga agttatgcac ctgttaaggc
     1501 taaactgaaa gaacagattt tggtgagtgc ggtcctccaa cccacttacc ttggttcaaa
     1561 gagttggtag ctcagcgaac cttgagaaaa ccaccgttgg tagcggtggt ttttctttat
     1621 ttatgagatg atgaatcaat cggtctatca agtcaacgaa cagctattcc gttactctag
     1681 atttcagtgc aatttatctc ttcaaatgta gcacctgaag tcagccccat acgatataag
     1741 ttgtaattct catgttagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
     1801 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg
     1861 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     1921 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     1981 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     2041 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     2101 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat gtccgcacca
     2161 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     2221 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     2281 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     2341 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     2401 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     2461 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     2521 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     2581 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     2641 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     2701 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     2761 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     2821 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     2881 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     2941 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     3001 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc
     3061 ctgcattagg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     3121 tgtagaaata attttgttta actttaataa ggagatatac catgggcagc agccatcatc
     3181 atcatcatca cagcagcggc ctggtgccgc gcggcagcgc tatgcagtac aaactgatcc
     3241 tgaacggtaa aaccctgaaa ggtgaaacca ccaccgaagc tgttgacgct gctaccgcgg
     3301 aaaaagtttt caaacagtac gctaacgaca acggtgttga cggtgaatgg acctacgacg
     3361 acgctaccaa aaccttcacc gttaccgaaa tggagaatct gtattttcag ggtggatcct
     3421 cagaacactc cacaggtata acccatcttc ctcctgaggt aatgctgtca attttcagct
     3481 atcttaatcc tcaagagtta tgtcgatgca gtcaagtaag catgaaatgg tctcagctga
     3541 caaaaacggg atcgctttgg aaacatcttt accctgttca ttgggccaga ggtgactggt
     3601 atagtggtcc cgcaactgaa cttgatactg aacctgatga tgaatgggtg aaaaatagga
     3661 aagatgaaag tcgtgctttt catgagtggg atgaagatgc tgacattgat gaatctgaag
     3721 agtctgcgga ggaatcaatt gctatcagca ttgcacaaat ggaaaaacgt ttactccatg
     3781 gcttaattca taacgttcta ccatatgttg gtacttctgt aaaaacctta gtattagcat
     3841 acagctctgc agtttccagc aaaatggtta ggcagatttt agagctttgt cctaacctgg
     3901 agcatctgga tcttacccag actgacattt cagattctgc atttgacagt tggtcttggc
     3961 ttggttgctg ccagagtctt cggcatcttg atctgtctgg ttgtgagaaa atcacagatg
     4021 tggccctaga gaagatttcc agagctcttg gaattctgac atctcatcaa agtggctttt
     4081 tgggttcggg aggtggacgt gtacttctgt ttctcagttt atctggatgt tatcagatca
     4141 cagaccatgg tctcagggtt ttgactctgg gaggagggct gccttatttg gagcacctta
     4201 atctctctgg ttgtcttact ataactggtg caggcctgca ggatttggtt tcagcatgtc
     4261 cttctctgaa tgatgaatac ttttactact gtgacaacat taacggtcct catgctgata
     4321 ccgccagtgg atgccagaat ttgcagtgtg gttttcgagc ctgctgccgc tctggcgaat
     4381 gagcggccgc ataatgctta agtcgaacag aaagtaatcg tattgtacac ggccgcataa
     4441 tcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc cccatcttag
     4501 tatattagtt aagtataaga aggagatata catatgcctt caattaagtt gcagagttct
     4561 gatggagaga tatttgaagt tgatgtggaa attgccaaac aatctgtgac tattaagacc
     4621 atgttggaag atttgggaat ggatgatgaa ggagatgatg acccagttcc tctaccaaat
     4681 gtgaatgcag caatattaaa aaaggtcatt cagtggtgca cccaccacaa ggatgaccct
     4741 cctcctcctg aagatgatga gaacaaagaa aagcgaacag atgatatccc tgtttgggac
     4801 caagaattcc tgaaagttga ccaaggaaca ctttttgaac tcattctggc tgcaaactac
     4861 ttagacatca aaggtttgct tgatgttaca tgcaagactg ttgccaatat gatcaagggg
     4921 aaaactcctg aggagattcg caagaccttc aatatcaaaa atgactttac tgaagaggag
     4981 gaagcccagg tacgcaaaga gaaccagtgg tgtgaagaga agtgaggtac cctcgagtct
     5041 ggtaaagaaa ccgctgctgc gaaatttgaa cgccagcaca tggactcgtc tactagcgca
     5101 gcttaattaa cctaggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     5161 tctaaacggg tcttgagggg ttttttgctg aaacctcagg catttgagaa gcacacggtc
     5221 acactgcttc cggtagtcaa taaaccggta aaccagcaat agacataagc ggctatttaa
     5281 cgaccctgcc ctgaaccgac gacaagctga cgaccgggtc tccgcaagtg gcacttttcg
     5341 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     5401 gctcatgaat taattc
//
