LOCUS       sequence-175082-        7232 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial expression vector pVP16 containing a mutant variant of 
            Arabidopsis thaliana PROTEOLYSIS 1 (PRT1) N-end rule E3 ubiquitin 
            ligase 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 7232)
  TITLE     PRT1 clones
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7232)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pVP16-tev-PRT1-V28A
FEATURES             Location/Qualifiers
     source          1..7232
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     559..578
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(598..675)
                     /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(759..781)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    867..1007
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     922..941
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1022..1039)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1193..1781)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1273..1292)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1952..2743,2744..2812))
                     /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     2575..2594
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2813..2917)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2985..3003
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(3027..3041,3042..3047,3048..3064,3065..3071)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    3047..3063
                     /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    3079..3095
                     /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             3114..3125
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3138..3161
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             3165..4262
                     /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     4236..4259
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    4332..4356
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4359..4379
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     protein_bind    complement(5614..5638)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      5706..5800
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             5844..6503
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(5910..5929)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      6568..6654
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(6747..7232,1..597))
                     /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 agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg
       61 gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
      121 aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca
      181 ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact
      241 gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc
      301 taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac
      361 aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg
      421 tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
      481 ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
      541 ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt
      601 caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgca
      661 ccattcgatg gtgtcggaat ttcgggcagc gttgggtcct ggccacgggt gcgcatgatc
      721 tagagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
      781 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
      841 tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga
      901 gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc
      961 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt
     1021 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     1081 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     1141 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     1201 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     1261 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     1321 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     1381 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     1441 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     1501 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     1561 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     1621 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     1681 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     1741 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     1801 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     1861 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     1921 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     1981 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     2041 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     2101 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     2161 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     2221 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     2281 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     2341 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     2401 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     2461 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     2521 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     2581 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     2641 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     2701 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     2761 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     2821 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     2881 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     2941 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     3001 ggccctttcg tcttcacctc gagaaatcat aaaaaattta tttgctttgt gagcggataa
     3061 caattataat agattcaatt gtgagcggat aacaatttca cacagaattc attaaagagg
     3121 agaaattaac catgggacat caccatcatc accatcacca ttcaaaaatc gaagaaggta
     3181 aactggtaat ctggattaac ggcgataaag gctataacgg tctcgctgaa gtcggtaaga
     3241 aattcgagaa agataccgga attaaagtca ccgttgagca tccggataaa ctggaagaga
     3301 aattcccaca ggttgcggca actggcgatg gccctgacat tatcttctgg gcacacgacc
     3361 gctttggtgg ctacgctcaa tctggcctgt tggctgaaat caccccggac aaagcgttcc
     3421 aggacaagct gtatccgttt acctgggatg ccgtacgtta caacggcaag ctgattgctt
     3481 acccgatcgc tgttgaagcg ttatcgctga tttataacaa agatctgctg ccgaacccgc
     3541 caaaaacctg ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa ggtaagagcg
     3601 cgctgatgtt caacctgcaa gaaccgtact tcacctggcc gctgattgct gctgacgggg
     3661 gttatgcgtt caagtatgaa aacggcaagt acgacattaa agacgtgggc gtggataacg
     3721 ctggcgcgaa agcgggtctg accttcctgg ttgacctgat taaaaacaaa cacatgaatg
     3781 cagacaccga ttactccatc gcagaagctg cctttaataa aggcgaaaca gcgatgacca
     3841 tcaacggccc gtgggcatgg tccaacatcg acaccagcaa agtgaattat ggtgtaacgg
     3901 tactgccgac cttcaagggt caaccatcca aaccgttcgt tggcgtgctg agcgcaggta
     3961 ttaacgccgc cagtccgaac aaagagctgg caaaagagtt cctcgaaaac tatctgctga
     4021 ctgatgaagg tctggaagcg gttaataaag acaaaccgct gggtgccgta gcgctgaagt
     4081 cttacgagga agagttggcg aaagatccac gtattgccgc cactatggaa aacgcccaga
     4141 aaggtgaaat catgccgaac atcccgcaga tgtccgcttt ctggtatgcc gtgcgtactg
     4201 cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga agccctgaaa gacgcgcaga
     4261 ctaattcgag ctcgaacaac aacaacaata acaataacaa caacctcggg atcgacgaaa
     4321 acctgtacct cacaagtttg tacaaaaaag caggcttaga gaatctttat tttcagggga
     4381 tggccgaaac tatgaaagat attacgatga agaacgatga atcacaggaa gaagaaatcc
     4441 ccgatcaatt tctctgctgc gcgtgcctgg aacttcttta caagccaatt gtgttatctt
     4501 gtggtcatct atcatgtttt tggtgcgtac ataagtccat gaatggcttt cgtgagtctc
     4561 attgtccgat atgtagagac ccgtatgttc actttccctc tgtgtgccag aagctttatt
     4621 ttctgttaaa gaagatgtac ccacttgctc ataagaagag agaagaacaa gttttaaagg
     4681 aagagcaaga acgagaatgt ttttctcctc agattgatct tgttttggat ttgtctgtgt
     4741 gtagtggaga ttctctcaat gtctctgata aacagaaggt ggaagagtgt tcgaatgcag
     4801 cgaacttatt atctagttca tcaagtagag gtgacattcc atgtatcccc aaaaatcaag
     4861 aacccacaga tgcaaaagct cttaatgttc atgaaaatga attacttaag gataacaaag
     4921 tcagtaagca gatttcgaaa gatgatttgc tctgttcagc atgtaaggag ctgcttgtac
     4981 gacccgtagt tctcaattgc ggacatgtgt attgtgaagg atgtgtagta gatatggctg
     5041 aagaaagcga aaagatcaaa tgtcaagagt gtaatgtttg tgacccaaga ggatttccaa
     5101 aagtttgttt gattcttgaa cagcttttgg aggaaaactt tcctgaagaa tacaattcaa
     5161 gaagcagtaa ggttcagaaa acgctcgccc ataatagcaa aggaaatatt caaagctatc
     5221 tcaaagaagg cccgtcctta tcaaacgaca ataacaatga tgatccctgg ttggcaaacc
     5281 ctggatcaaa tgttcacttt ggagctggtt gtgattcttg tggggtgtat ccaatcatag
     5341 gggatcgata cagatgcaaa gactgcaagg aggaaattgg gtatgacctt tgcaaagact
     5401 gttacgagac tccttcgaaa gttccaggga gattcaacca gcaacacact cctgaccaca
     5461 ggcttgagct tgcacggtct cctcaggttc tgatcaattt caattctatc ggtatccttc
     5521 tcggacccgt tatctcaaat gaaggcatgg atacagatga aggcgaggaa gggcctcctg
     5581 gttcttctaa tgagtcatca agcacagaat gatacccagc tttcttgtac aaagtggtga
     5641 tggcgcgcct gtaggacgtc gacggtacca tcgatacgcg ttcgaagctt aattagctga
     5701 gcttggactc ctgttgatag atccagtaat gacctcagaa ctccatctgg atttgttcag
     5761 aacgctcggt tgccgccggg cgttttttat tggtgagaat ccaagctagc ttggcgagat
     5821 tttcaggagc taaggaagct aaaatggaga aaaaaatcac tggatatacc accgttgata
     5881 tatcccaatg gcatcgtaaa gaacattttg aggcatttca gtcagttgct caatgtacct
     5941 ataaccagac cgttcagctg gatattacgg cctttttaaa gaccgtaaag aaaaataagc
     6001 acaagtttta tccggccttt attcacattc ttgcccgcct gatgaatgct catccggaat
     6061 ttcgtatggc aatgaaagac ggtgagctgg tgatatggga tagtgttcac ccttgttaca
     6121 ccgttttcca tgagcaaact gaaacgtttt catcgctctg gagtgaatac cacgacgatt
     6181 tccggcagtt tctacacata tattcgcaag atgtggcgtg ttacggtgaa aacctggcct
     6241 atttccctaa agggtttatt gagaatatgt ttttcgtctc agccaatccc tgggtgagtt
     6301 tcaccagttt tgatttaaac gtggccaata tggacaactt cttcgccccc gttttcacca
     6361 tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt caggttcatc
     6421 atgccgtttg tgatggcttc catgtcggca gaatgcttaa tgaattacaa cagtactgcg
     6481 atgagtggca gggcggggcg taattttttt aaggcagtta ttggtgccct taaacgcctg
     6541 gggtaatgac tctctagctt gaggcatcaa ataaaacgaa aggctcagtc gaaagactgg
     6601 gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccc
     6661 tctagattac gtgcagtcga tgataagctg tcaaacatga gaattgtgcc taatgagtga
     6721 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     6781 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     6841 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     6901 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     6961 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     7021 accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     7081 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     7141 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     7201 atttgattgc gagtgagata tttatgccag cc
//
