LOCUS       sequence-174593-        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-Tyr-eK-Flv
FEATURES             Location/Qualifiers
     source          1..6662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     921..940
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(960..1037)
                     /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     complement(1121..1143)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1229..1369
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1284..1303
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1384..1401)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1555..2143)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1635..1654)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2314..3105,3106..3174))
                     /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     2937..2956
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3175..3279)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3347..3365
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(3389..3403,3404..3409,3410..3426,3427..3433)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    3409..3425
                     /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)."
     protein_bind    3441..3457
                     /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             3476..3487
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3500..3523
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             3527..4624
                     /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     4598..4621
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    4694..4718
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4865..4954
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     protein_bind    complement(5406..5430)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      5498..5592
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             5636..6295
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(5702..5721)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      6360..6446
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(6539..6662,1..959))
                     /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"
ORIGIN
        1 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
       61 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
      121 gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg
      181 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
      241 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
      301 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
      361 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
      421 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
      481 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
      541 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
      601 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
      661 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
      721 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
      781 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
      841 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
      901 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
      961 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     1021 caccattcga tggtgtcgga atttcgggca gcgttgggtc ctggccacgg gtgcgcatga
     1081 tctagagctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
     1141 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
     1201 cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg
     1261 gagtgtatac tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat
     1321 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc
     1381 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     1441 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     1501 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     1561 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     1621 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     1681 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     1741 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     1801 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     1861 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     1921 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     1981 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     2041 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     2101 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     2161 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     2221 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     2281 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     2341 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     2401 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     2461 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     2521 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     2581 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     2641 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     2701 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     2761 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     2821 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     2881 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     2941 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     3001 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     3061 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     3121 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     3181 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     3241 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     3301 acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac
     3361 gaggcccttt cgtcttcacc tcgagaaatc ataaaaaatt tatttgcttt gtgagcggat
     3421 aacaattata atagattcaa ttgtgagcgg ataacaattt cacacagaat tcattaaaga
     3481 ggagaaatta accatgggac atcaccatca tcaccatcac cattcaaaaa tcgaagaagg
     3541 taaactggta atctggatta acggcgataa aggctataac ggtctcgctg aagtcggtaa
     3601 gaaattcgag aaagataccg gaattaaagt caccgttgag catccggata aactggaaga
     3661 gaaattccca caggttgcgg caactggcga tggccctgac attatcttct gggcacacga
     3721 ccgctttggt ggctacgctc aatctggcct gttggctgaa atcaccccgg acaaagcgtt
     3781 ccaggacaag ctgtatccgt ttacctggga tgccgtacgt tacaacggca agctgattgc
     3841 ttacccgatc gctgttgaag cgttatcgct gatttataac aaagatctgc tgccgaaccc
     3901 gccaaaaacc tgggaagaga tcccggcgct ggataaagaa ctgaaagcga aaggtaagag
     3961 cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg ccgctgattg ctgctgacgg
     4021 gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa
     4081 cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg attaaaaaca aacacatgaa
     4141 tgcagacacc gattactcca tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac
     4201 catcaacggc ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt atggtgtaac
     4261 ggtactgccg accttcaagg gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg
     4321 tattaacgcc gccagtccga acaaagagct ggcaaaagag ttcctcgaaa actatctgct
     4381 gactgatgaa ggtctggaag cggttaataa agacaaaccg ctgggtgccg tagcgctgaa
     4441 gtcttacgag gaagagttgg cgaaagatcc acgtattgcc gccactatgg aaaacgccca
     4501 gaaaggtgaa atcatgccga acatcccgca gatgtccgct ttctggtatg ccgtgcgtac
     4561 tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat gaagccctga aagacgcgca
     4621 gactaattcg agctcgaaca acaacaacaa taacaataac aacaacctcg ggatcgacga
     4681 aaacctgtac ctcacaagtt tgtacaaaaa agcaggctta gaaaacctgt attttcagta
     4741 ccacggatct ggagcttggc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc
     4801 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agggaatggg
     4861 atcctaccca tacgatgttc ctgactatgc gggctatccc tatgacgtcc cggactatgc
     4921 aggatcctat ccatacgacg ttccagatta cgctggtgct gcagatatca ctcttatcag
     4981 cggcagcacc ctcggcggtg ccgaatatgt agcagaacac ctggctgaaa agctggaaga
     5041 ggcgggtttt accaccgaaa cgctgcacgg tccgctgttg gaagatttat ctgcctcggg
     5101 gatctggctg attatcagct ccacccacgg tgccggagat attccggaca acctttctcc
     5161 tttctatgaa gcattgcagg aacagaagcc cgatctttct gcggtccgct ttggcgcaat
     5221 cggtattggt agtcgtgaat atgacacctt ttgtggggct atcgataaac tcgaggcaga
     5281 actcaaagat tccggtgcaa aacagacagg cgaaacactg aagatcaaca ttcttgatca
     5341 cgacattccg gaagatccgg cagaagaatg gctgggatcg tggattaatt tactcaaata
     5401 atgataccca gctttcttgt acaaagtggt gatggcgcgc ctgtaggacg tcgacggtac
     5461 catcgatacg cgttcgaagc ttaattagct gagcttggac tcctgttgat agatccagta
     5521 atgacctcag aactccatct ggatttgttc agaacgctcg gttgccgccg ggcgtttttt
     5581 attggtgaga atccaagcta gcttggcgag attttcagga gctaaggaag ctaaaatgga
     5641 gaaaaaaatc actggatata ccaccgttga tatatcccaa tggcatcgta aagaacattt
     5701 tgaggcattt cagtcagttg ctcaatgtac ctataaccag accgttcagc tggatattac
     5761 ggccttttta aagaccgtaa agaaaaataa gcacaagttt tatccggcct ttattcacat
     5821 tcttgcccgc ctgatgaatg ctcatccgga atttcgtatg gcaatgaaag acggtgagct
     5881 ggtgatatgg gatagtgttc acccttgtta caccgttttc catgagcaaa ctgaaacgtt
     5941 ttcatcgctc tggagtgaat accacgacga tttccggcag tttctacaca tatattcgca
     6001 agatgtggcg tgttacggtg aaaacctggc ctatttccct aaagggttta ttgagaatat
     6061 gtttttcgtc tcagccaatc cctgggtgag tttcaccagt tttgatttaa acgtggccaa
     6121 tatggacaac ttcttcgccc ccgttttcac catgggcaaa tattatacgc aaggcgacaa
     6181 ggtgctgatg ccgctggcga ttcaggttca tcatgccgtt tgtgatggct tccatgtcgg
     6241 cagaatgctt aatgaattac aacagtactg cgatgagtgg cagggcgggg cgtaattttt
     6301 ttaaggcagt tattggtgcc cttaaacgcc tggggtaatg actctctagc ttgaggcatc
     6361 aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg
     6421 tgaacgctct cctgagtagg acaaatccgc cctctagatt acgtgcagtc gatgataagc
     6481 tgtcaaacat gagaattgtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     6541 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     6601 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     6661 cg
//
