LOCUS       sequence-175081-        7232 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial expression vector pVP16 containing 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  JOURNAL   Unpublished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            "Unpublished"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
FEATURES             Location/Qualifiers
     source          1..7232
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     555..574
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(594..671)
                     /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(755..777)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    863..1003
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     918..937
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1018..1035)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1189..1777)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1269..1288)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1948..2739,2740..2808))
                     /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     2571..2590
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2809..2913)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2981..2999
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(3023..3037,3038..3043,3044..3060,3061..3067)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    3043..3059
                     /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    3075..3091
                     /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             3110..3121
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3134..3157
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             3161..4258
                     /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     4232..4255
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    4328..4352
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4355..4375
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     protein_bind    complement(5610..5634)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      5702..5796
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             5840..6499
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(5906..5925)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      6564..6650
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(6743..7232,1..593))
                     /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 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
       61 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
      121 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
      181 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
      241 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
      301 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
      361 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
      421 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
      481 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
      541 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
      601 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcaccat
      661 tcgatggtgt cggaatttcg ggcagcgttg ggtcctggcc acgggtgcgc atgatctaga
      721 gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
      781 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
      841 cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt
      901 atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg
      961 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc
     1021 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     1081 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     1141 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     1201 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     1261 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     1321 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     1381 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     1441 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     1501 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     1561 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     1621 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     1681 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     1741 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     1801 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     1861 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     1921 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     1981 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     2041 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     2101 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     2161 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     2221 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     2281 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     2341 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     2401 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     2461 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     2521 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     2581 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     2641 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     2701 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     2761 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     2821 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     2881 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     2941 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     3001 ctttcgtctt cacctcgaga aatcataaaa aatttatttg ctttgtgagc ggataacaat
     3061 tataatagat tcaattgtga gcggataaca atttcacaca gaattcatta aagaggagaa
     3121 attaaccatg ggacatcacc atcatcacca tcaccattca aaaatcgaag aaggtaaact
     3181 ggtaatctgg attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt
     3241 cgagaaagat accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt
     3301 cccacaggtt gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt
     3361 tggtggctac gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga
     3421 caagctgtat ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc
     3481 gatcgctgtt gaagcgttat cgctgattta taacaaagat ctgctgccga acccgccaaa
     3541 aacctgggaa gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct
     3601 gatgttcaac ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta
     3661 tgcgttcaag tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg
     3721 cgcgaaagcg ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga
     3781 caccgattac tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa
     3841 cggcccgtgg gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact
     3901 gccgaccttc aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa
     3961 cgccgccagt ccgaacaaag agctggcaaa agagttcctc gaaaactatc tgctgactga
     4021 tgaaggtctg gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta
     4081 cgaggaagag ttggcgaaag atccacgtat tgccgccact atggaaaacg cccagaaagg
     4141 tgaaatcatg ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt
     4201 gatcaacgcc gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg cgcagactaa
     4261 ttcgagctcg aacaacaaca acaataacaa taacaacaac ctcgggatcg acgaaaacct
     4321 gtacctcaca agtttgtaca aaaaagcagg cttagagaat ctttattttc aggggatggc
     4381 cgaaactatg aaagatatta cgatgaagaa cgatgaatca caggaagaag aaatccccga
     4441 tcaatttctc tgctgcgttt gcctggaact tctttacaag ccaattgtgt tatcttgtgg
     4501 tcatctatca tgtttttggt gcgtacataa gtccatgaat ggctttcgtg agtctcattg
     4561 tccgatatgt agagacccgt atgttcactt tccctctgtg tgccagaagc tttattttct
     4621 gttaaagaag atgtacccac ttgctcataa gaagagagaa gaacaagttt taaaggaaga
     4681 gcaagaacga gaatgttttt ctcctcagat tgatcttgtt ttggatttgt ctgtgtgtag
     4741 tggagattct ctcaatgtct ctgataaaca gaaggtggaa gagtgttcga atgcagcgaa
     4801 cttattatct agttcatcaa gtagaggtga cattccatgt atccccaaaa atcaagaacc
     4861 cacagatgca aaagctctta atgttcatga aaatgaatta cttaaggata acaaagtcag
     4921 taagcagatt tcgaaagatg atttgctctg ttcagcatgt aaggagctgc ttgtacgacc
     4981 cgtagttctc aattgcggac atgtgtattg tgaaggatgt gtagtagata tggctgaaga
     5041 aagcgaaaag atcaaatgtc aagagtgtaa tgtttgtgac ccaagaggat ttccaaaagt
     5101 ttgtttgatt cttgaacagc ttttggagga aaactttcct gaagaataca attcaagaag
     5161 cagtaaggtt cagaaaacgc tcgcccataa tagcaaagga aatattcaaa gctatctcaa
     5221 agaaggcccg tccttatcaa acgacaataa caatgatgat ccctggttgg caaaccctgg
     5281 atcaaatgtt cactttggag ctggttgtga ttcttgtggg gtgtatccaa tcatagggga
     5341 tcgatacaga tgcaaagact gcaaggagga aattgggtat gacctttgca aagactgtta
     5401 cgagactcct tcgaaagttc cagggagatt caaccagcaa cacactcctg accacaggct
     5461 tgagcttgca cggtctcctc aggttctgat caatttcaat tctatcggta tccttctcgg
     5521 acccgttatc tcaaatgaag gcatggatac agatgaaggc gaggaagggc ctcctggttc
     5581 ttctaatgag tcatcaagca cagaatgata cccagctttc ttgtacaaag tggtgatggc
     5641 gcgcctgtag gacgtcgacg gtaccatcga tacgcgttcg aagcttaatt agctgagctt
     5701 ggactcctgt tgatagatcc agtaatgacc tcagaactcc atctggattt gttcagaacg
     5761 ctcggttgcc gccgggcgtt ttttattggt gagaatccaa gctagcttgg cgagattttc
     5821 aggagctaag gaagctaaaa tggagaaaaa aatcactgga tataccaccg ttgatatatc
     5881 ccaatggcat cgtaaagaac attttgaggc atttcagtca gttgctcaat gtacctataa
     5941 ccagaccgtt cagctggata ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa
     6001 gttttatccg gcctttattc acattcttgc ccgcctgatg aatgctcatc cggaatttcg
     6061 tatggcaatg aaagacggtg agctggtgat atgggatagt gttcaccctt gttacaccgt
     6121 tttccatgag caaactgaaa cgttttcatc gctctggagt gaataccacg acgatttccg
     6181 gcagtttcta cacatatatt cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt
     6241 ccctaaaggg tttattgaga atatgttttt cgtctcagcc aatccctggg tgagtttcac
     6301 cagttttgat ttaaacgtgg ccaatatgga caacttcttc gcccccgttt tcaccatggg
     6361 caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgattcagg ttcatcatgc
     6421 cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa ttacaacagt actgcgatga
     6481 gtggcagggc ggggcgtaat ttttttaagg cagttattgg tgcccttaaa cgcctggggt
     6541 aatgactctc tagcttgagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct
     6601 ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccctcta
     6661 gattacgtgc agtcgatgat aagctgtcaa acatgagaat tgtgcctaat gagtgagcta
     6721 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     6781 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     6841 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     6901 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     6961 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     7021 agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     7081 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     7141 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     7201 gattgcgagt gagatattta tgccagccag cc
//
