LOCUS       sequence-406419-        4993 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of VHL (54-213) for production of VCB complexes in 
            conjunction with plasmid pIVM_26 (EloB 1-104, EloC 17-112).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4993)
  AUTHORS   Cardote TAF, Gadd MS, Ciulli A
  TITLE     Crystal Structure of the Cul2-Rbx1-EloBC-VHL Ubiquitin Ligase 
            Complex.
  JOURNAL   Structure. 2017 Jun 6;25(6):901-911.e3. doi: 
            10.1016/j.str.2017.04.009.
  PUBMED    28591624
REFERENCE   2  (bases 1 to 4993)
  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.str.2017.04"; journalName: "Structure"; date: "2017-06-6-
            6"; volume: "25"; issue: "6"; pages: "901-911.e3"
COMMENT     Sequence Label: pIVM_02
FEATURES             Location/Qualifiers
     source          1..4993
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     154..171
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     242..261
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        242..260
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    260..284
                     /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)."
     RBS             297..319
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             339..356
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             390..407
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             414..434
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     promoter        complement(984..1002)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(985..1002)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1060..1078)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1074..1121
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1357..1378)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1528..2610)
                     /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     2572..2591
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2611..2688)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     2831..2850
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3004..3023)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3123..3145
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3183..3201)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3269..3373
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3374..3442,3443..4234)
                     /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(3592..3611)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4405..4993
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4894..4913
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
       61 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
      121 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
      181 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctga
      241 ttaatacgac tcactatagg gaattgtgag cggataacaa ttcctctaga aataattttg
      301 tttaacttta agaaggagat atatccatga acaccattca tcaccatcac catcacaaca
      361 ctagtggatc tggtggtggt ggcggccgcc tggttccgag gggatccatg tctgaaaacc
      421 tgtacttcca gggttccatg gaggccgggc ggccgcggcc cgtgctgcgc tcggtgaact
      481 cgcgcgagcc ctcccaggtc atcttctgca atcgcagtcc gcgcgtcgtg ctgcccgtat
      541 ggctcaactt cgacggcgag ccgcagccct acccaacgct gccgcctggc acgggccgcc
      601 gcatccacag ctaccgaggt cacctttggc tcttcagaga tgcagggaca cacgatgggc
      661 ttctggttaa ccaaactgaa ttatttgtgc catctctcaa tgttgacgga cagcctattt
      721 ttgccaatat cacactgcca gtgtatactc tgaaagagcg atgcctccag gttgtccgga
      781 gcctagtcaa gcctgagaat tacaggagac tggacatcgt caggtcgctc tacgaagatc
      841 tggaagacca cccaaatgtg cagaaagacc tggagcggct gacacaggag cgcattgcac
      901 atcaacggat gggagattga gaattcgtcg acctcgaggg atccgggccc tctagatgcg
      961 gccgcatgca taagcttgag tattctatag tgtcacctaa atcccagctt gatccggctg
     1021 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat
     1081 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
     1141 ccggataacc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     1201 cagcctgaat ggcgaatgga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     1261 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     1321 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     1381 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     1441 cagcttgcat gcctgcaggt cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     1501 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     1561 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     1621 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     1681 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     1741 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     1801 taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     1861 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     1921 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     1981 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     2041 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     2101 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     2161 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     2221 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     2281 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     2341 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     2401 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     2461 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     2521 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     2581 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     2641 gcgctatcat gccataccgc gaaaggtttt gcaccattcg atggtgtcgg gtaccgagct
     2701 cgaattgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     2761 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     2821 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     2881 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcct
     2941 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct
     3001 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
     3061 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     3121 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa
     3181 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     3241 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     3301 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     3361 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     3421 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     3481 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     3541 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     3601 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     3661 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     3721 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     3781 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     3841 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     3901 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     3961 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     4021 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     4081 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     4141 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     4201 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     4261 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     4321 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     4381 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     4441 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     4501 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     4561 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     4621 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     4681 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     4741 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     4801 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     4861 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     4921 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     4981 gagcctatgg aaa
//
