LOCUS       sequence-174594-        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-Ile-eK-Flv
FEATURES             Location/Qualifiers
     source          1..6662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     963..982
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1002..1079)
                     /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(1163..1185)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1271..1411
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1326..1345
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1426..1443)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1597..2185)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1677..1696)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2356..3147,3148..3216))
                     /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     2979..2998
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3217..3321)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3389..3407
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(3431..3445,3446..3451,3452..3468,3469..3475)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    3451..3467
                     /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    3483..3499
                     /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             3518..3529
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3542..3565
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             3569..4666
                     /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     4640..4663
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    4736..4760
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4907..4996
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     protein_bind    complement(5448..5472)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      5540..5634
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             5678..6337
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(5744..5763)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      6402..6488
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(6581..6662,1..1001))
                     /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 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
       61 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
      121 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
      181 tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc
      241 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
      301 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
      361 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
      421 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
      481 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
      541 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
      601 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
      661 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
      721 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
      781 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
      841 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
      901 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
      961 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     1021 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcaccattc gatggtgtcg
     1081 gaatttcggg cagcgttggg tcctggccac gggtgcgcat gatctagagc tgcctcgcgc
     1141 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     1201 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     1261 ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa
     1321 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca
     1381 cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc
     1441 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     1501 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     1561 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     1621 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     1681 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     1741 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     1801 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     1861 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     1921 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     1981 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     2041 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     2101 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     2161 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     2221 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     2281 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     2341 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     2401 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     2461 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     2521 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     2581 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     2641 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     2701 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     2761 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     2821 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     2881 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     2941 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     3001 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     3061 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     3121 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     3181 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     3241 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     3301 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac
     3361 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtcttca
     3421 cctcgagaaa tcataaaaaa tttatttgct ttgtgagcgg ataacaatta taatagattc
     3481 aattgtgagc ggataacaat ttcacacaga attcattaaa gaggagaaat taaccatggg
     3541 acatcaccat catcaccatc accattcaaa aatcgaagaa ggtaaactgg taatctggat
     3601 taacggcgat aaaggctata acggtctcgc tgaagtcggt aagaaattcg agaaagatac
     3661 cggaattaaa gtcaccgttg agcatccgga taaactggaa gagaaattcc cacaggttgc
     3721 ggcaactggc gatggccctg acattatctt ctgggcacac gaccgctttg gtggctacgc
     3781 tcaatctggc ctgttggctg aaatcacccc ggacaaagcg ttccaggaca agctgtatcc
     3841 gtttacctgg gatgccgtac gttacaacgg caagctgatt gcttacccga tcgctgttga
     3901 agcgttatcg ctgatttata acaaagatct gctgccgaac ccgccaaaaa cctgggaaga
     3961 gatcccggcg ctggataaag aactgaaagc gaaaggtaag agcgcgctga tgttcaacct
     4021 gcaagaaccg tacttcacct ggccgctgat tgctgctgac gggggttatg cgttcaagta
     4081 tgaaaacggc aagtacgaca ttaaagacgt gggcgtggat aacgctggcg cgaaagcggg
     4141 tctgaccttc ctggttgacc tgattaaaaa caaacacatg aatgcagaca ccgattactc
     4201 catcgcagaa gctgccttta ataaaggcga aacagcgatg accatcaacg gcccgtgggc
     4261 atggtccaac atcgacacca gcaaagtgaa ttatggtgta acggtactgc cgaccttcaa
     4321 gggtcaacca tccaaaccgt tcgttggcgt gctgagcgca ggtattaacg ccgccagtcc
     4381 gaacaaagag ctggcaaaag agttcctcga aaactatctg ctgactgatg aaggtctgga
     4441 agcggttaat aaagacaaac cgctgggtgc cgtagcgctg aagtcttacg aggaagagtt
     4501 ggcgaaagat ccacgtattg ccgccactat ggaaaacgcc cagaaaggtg aaatcatgcc
     4561 gaacatcccg cagatgtccg ctttctggta tgccgtgcgt actgcggtga tcaacgccgc
     4621 cagcggtcgt cagactgtcg atgaagccct gaaagacgcg cagactaatt cgagctcgaa
     4681 caacaacaac aataacaata acaacaacct cgggatcgac gaaaacctgt acctcacaag
     4741 tttgtacaaa aaagcaggct tagaaaacct gtattttcag attcacggat ctggagcttg
     4801 gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc
     4861 ctctccccgc gcgttggccg attcattaat gcagggaatg ggatcctacc catacgatgt
     4921 tcctgactat gcgggctatc cctatgacgt cccggactat gcaggatcct atccatacga
     4981 cgttccagat tacgctggtg ctgcagatat cactcttatc agcggcagca ccctcggcgg
     5041 tgccgaatat gtagcagaac acctggctga aaagctggaa gaggcgggtt ttaccaccga
     5101 aacgctgcac ggtccgctgt tggaagattt atctgcctcg gggatctggc tgattatcag
     5161 ctccacccac ggtgccggag atattccgga caacctttct cctttctatg aagcattgca
     5221 ggaacagaag cccgatcttt ctgcggtccg ctttggcgca atcggtattg gtagtcgtga
     5281 atatgacacc ttttgtgggg ctatcgataa actcgaggca gaactcaaag attccggtgc
     5341 aaaacagaca ggcgaaacac tgaagatcaa cattcttgat cacgacattc cggaagatcc
     5401 ggcagaagaa tggctgggat cgtggattaa tttactcaaa taatgatacc cagctttctt
     5461 gtacaaagtg gtgatggcgc gcctgtagga cgtcgacggt accatcgata cgcgttcgaa
     5521 gcttaattag ctgagcttgg actcctgttg atagatccag taatgacctc agaactccat
     5581 ctggatttgt tcagaacgct cggttgccgc cgggcgtttt ttattggtga gaatccaagc
     5641 tagcttggcg agattttcag gagctaagga agctaaaatg gagaaaaaaa tcactggata
     5701 taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat ttcagtcagt
     5761 tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt taaagaccgt
     5821 aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc gcctgatgaa
     5881 tgctcatccg gaatttcgta tggcaatgaa agacggtgag ctggtgatat gggatagtgt
     5941 tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc tctggagtga
     6001 ataccacgac gatttccggc agtttctaca catatattcg caagatgtgg cgtgttacgg
     6061 tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg tctcagccaa
     6121 tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca acttcttcgc
     6181 ccccgttttc accatgggca aatattatac gcaaggcgac aaggtgctga tgccgctggc
     6241 gattcaggtt catcatgccg tttgtgatgg cttccatgtc ggcagaatgc ttaatgaatt
     6301 acaacagtac tgcgatgagt ggcagggcgg ggcgtaattt ttttaaggca gttattggtg
     6361 cccttaaacg cctggggtaa tgactctcta gcttgaggca tcaaataaaa cgaaaggctc
     6421 agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta
     6481 ggacaaatcc gccctctaga ttacgtgcag tcgatgataa gctgtcaaac atgagaattg
     6541 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     6601 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     6661 gc
//
