LOCUS       sequence-174595-        6662 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial expression vector pVP16 containing a derivative of E. coli
            lacZ fused with E. coli flavin binding protein MioC to be used in 
            N-end rule in vitro ubiquitination studies.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6662)
  AUTHORS   Mot AC, Prell E, Klecker M, Naumann C, Faden F, Westermann B, 
            Dissmeyer N
  TITLE     Real-time detection of N-end rule-mediated ubiquitination via 
            fluorescently labeled substrate probes.
  JOURNAL   New Phytol. 2017 Mar 9. doi: 10.1111/nph.14497.
  PUBMED    28277608
REFERENCE   2  (bases 1 to 6662)
  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: "New Phytol.
            2017 Mar 9. doi: 10.1111/nph.14497."
COMMENT     Sequence Label: His8:MBP-tev-Ala-eK-Flv
FEATURES             Location/Qualifiers
     source          1..6662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(71..91)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQA"
     protein_bind    complement(94..118)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(188..1285)
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     complement(191..214)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             complement(1289..1312)
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     RBS             1325..1336
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    1355..1371
                     /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(1379..1385,1386..1402,1403..1408,
                     1409..1423))
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    1387..1403
                     /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(1447..1465)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1533..1637
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1638..1706,1707..2498)
                     /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(1856..1875)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2669..3257
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3158..3177
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3411..3428
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3443..3583
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3509..3528)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3669..3691
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        3775..3852
                     /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."
     CDS             3853..4935
                     /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     complement(3872..3891)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     terminator      complement(5028..5114)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(5179..5838)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5753..5772
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      complement(5882..5976)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     protein_bind    6044..6068
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(6520..6609)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
ORIGIN
        1 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agccaagctc
       61 cagatccgtg agcctgaaaa tacaggtttt ctaagcctgc ttttttgtac aaacttgtga
      121 ggtacaggtt ttcgtcgatc ccgaggttgt tgttattgtt attgttgttg ttgttcgagc
      181 tcgaattagt ctgcgcgtct ttcagggctt catcgacagt ctgacgaccg ctggcggcgt
      241 tgatcaccgc agtacgcacg gcataccaga aagcggacat ctgcgggatg ttcggcatga
      301 tttcaccttt ctgggcgttt tccatagtgg cggcaatacg tggatctttc gccaactctt
      361 cctcgtaaga cttcagcgct acggcaccca gcggtttgtc tttattaacc gcttccagac
      421 cttcatcagt cagcagatag ttttcgagga actcttttgc cagctctttg ttcggactgg
      481 cggcgttaat acctgcgctc agcacgccaa cgaacggttt ggatggttga cccttgaagg
      541 tcggcagtac cgttacacca taattcactt tgctggtgtc gatgttggac catgcccacg
      601 ggccgttgat ggtcatcgct gtttcgcctt tattaaaggc agcttctgcg atggagtaat
      661 cggtgtctgc attcatgtgt ttgtttttaa tcaggtcaac caggaaggtc agacccgctt
      721 tcgcgccagc gttatccacg cccacgtctt taatgtcgta cttgccgttt tcatacttga
      781 acgcataacc cccgtcagca gcaatcagcg gccaggtgaa gtacggttct tgcaggttga
      841 acatcagcgc gctcttacct ttcgctttca gttctttatc cagcgccggg atctcttccc
      901 aggtttttgg cgggttcggc agcagatctt tgttataaat cagcgataac gcttcaacag
      961 cgatcgggta agcaatcagc ttgccgttgt aacgtacggc atcccaggta aacggataca
     1021 gcttgtcctg gaacgctttg tccggggtga tttcagccaa caggccagat tgagcgtagc
     1081 caccaaagcg gtcgtgtgcc cagaagataa tgtcagggcc atcgccagtt gccgcaacct
     1141 gtgggaattt ctcttccagt ttatccggat gctcaacggt gactttaatt ccggtatctt
     1201 tctcgaattt cttaccgact tcagcgagac cgttatagcc tttatcgccg ttaatccaga
     1261 ttaccagttt accttcttcg atttttgaat ggtgatggtg atgatggtga tgtcccatgg
     1321 ttaatttctc ctctttaatg aattctgtgt gaaattgtta tccgctcaca attgaatcta
     1381 ttataattgt tatccgctca caaagcaaat aaatttttta tgatttctcg aggtgaagac
     1441 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     1501 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     1561 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     1621 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     1681 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     1741 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     1801 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     1861 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     1921 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     1981 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     2041 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     2101 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     2161 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     2221 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     2281 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     2341 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     2401 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     2461 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     2521 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     2581 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     2641 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     2701 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     2761 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     2821 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     2881 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     2941 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     3001 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     3061 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     3121 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     3181 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     3241 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     3301 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     3361 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     3421 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     3481 tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     3541 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     3601 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     3661 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     3721 gcagctctag atcatgcgca cccgtggcca ggacccaacg ctgcccgaaa ttccgacacc
     3781 atcgaatggt gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc
     3841 agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct
     3901 tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa
     3961 aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg
     4021 gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg
     4081 tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg
     4141 tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg
     4201 caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg
     4261 gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc
     4321 aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca
     4381 ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg
     4441 cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg
     4501 gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc
     4561 atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc
     4621 attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc
     4681 gaagacagct catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg
     4741 gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat
     4801 cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc
     4861 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     4921 gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcacaatt
     4981 ctcatgtttg acagcttatc atcgactgca cgtaatctag agggcggatt tgtcctactc
     5041 aggagagcgt tcaccgacaa acaacagata aaacgaaagg cccagtcttt cgactgagcc
     5101 tttcgtttta tttgatgcct caagctagag agtcattacc ccaggcgttt aagggcacca
     5161 ataactgcct taaaaaaatt acgccccgcc ctgccactca tcgcagtact gttgtaattc
     5221 attaagcatt ctgccgacat ggaagccatc acaaacggca tgatgaacct gaatcgccag
     5281 cggcatcagc accttgtcgc cttgcgtata atatttgccc atggtgaaaa cgggggcgaa
     5341 gaagttgtcc atattggcca cgtttaaatc aaaactggtg aaactcaccc agggattggc
     5401 tgagacgaaa aacatattct caataaaccc tttagggaaa taggccaggt tttcaccgta
     5461 acacgccaca tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt ggtattcact
     5521 ccagagcgat gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag ggtgaacact
     5581 atcccatatc accagctcac cgtctttcat tgccatacga aattccggat gagcattcat
     5641 caggcgggca agaatgtgaa taaaggccgg ataaaacttg tgcttatttt tctttacggt
     5701 ctttaaaaag gccgtaatat ccagctgaac ggtctggtta taggtacatt gagcaactga
     5761 ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat atatcaacgg tggtatatcc
     5821 agtgattttt ttctccattt tagcttcctt agctcctgaa aatctcgcca agctagcttg
     5881 gattctcacc aataaaaaac gcccggcggc aaccgagcgt tctgaacaaa tccagatgga
     5941 gttctgaggt cattactgga tctatcaaca ggagtccaag ctcagctaat taagcttcga
     6001 acgcgtatcg atggtaccgt cgacgtccta caggcgcgcc atcaccactt tgtacaagaa
     6061 agctgggtat cattatttga gtaaattaat ccacgatccc agccattctt ctgccggatc
     6121 ttccggaatg tcgtgatcaa gaatgttgat cttcagtgtt tcgcctgtct gttttgcacc
     6181 ggaatctttg agttctgcct cgagtttatc gatagcccca caaaaggtgt catattcacg
     6241 actaccaata ccgattgcgc caaagcggac cgcagaaaga tcgggcttct gttcctgcaa
     6301 tgcttcatag aaaggagaaa ggttgtccgg aatatctccg gcaccgtggg tggagctgat
     6361 aatcagccag atccccgagg cagataaatc ttccaacagc ggaccgtgca gcgtttcggt
     6421 ggtaaaaccc gcctcttcca gcttttcagc caggtgttct gctacatatt cggcaccgcc
     6481 gagggtgctg ccgctgataa gagtgatatc tgcagcacca gcgtaatctg gaacgtcgta
     6541 tggataggat cctgcatagt ccgggacgtc atagggatag cccgcatagt caggaacatc
     6601 gtatgggtag gatcccattc cctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     6661 tt
//
