LOCUS       sequence-175084-        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  JOURNAL   Unpublished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            "Unpublished"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-C195A
FEATURES             Location/Qualifiers
     source          1..7232
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     578..597
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(617..694)
                     /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(778..800)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    886..1026
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     941..960
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1041..1058)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1212..1800)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1292..1311)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1971..2762,2763..2831))
                     /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     2594..2613
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2832..2936)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3004..3022
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(3046..3060,3061..3066,3067..3083,3084..3090)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    3066..3082
                     /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    3098..3114
                     /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             3133..3144
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3157..3180
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             3184..4281
                     /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     4255..4278
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    4351..4375
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4378..4398
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     protein_bind    complement(5633..5657)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      5725..5819
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             5863..6522
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(5929..5948)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      6587..6673
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(6766..7232,1..616))
                     /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 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
       61 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
      121 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
      181 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
      241 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
      301 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
      361 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
      421 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
      481 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
      541 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
      601 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
      661 taccgcgaaa ggttttgcac cattcgatgg tgtcggaatt tcgggcagcg ttgggtcctg
      721 gccacgggtg cgcatgatct agagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct
      781 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag
      841 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca
      901 gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta
      961 ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     1021 atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     1081 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     1141 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     1201 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     1261 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     1321 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     1381 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     1441 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     1501 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     1561 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     1621 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg
     1681 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     1741 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     1801 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     1861 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     1921 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     1981 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     2041 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     2101 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     2161 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     2221 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     2281 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     2341 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     2401 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     2461 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     2521 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     2581 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     2641 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     2701 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     2761 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     2821 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     2881 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     2941 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     3001 aaaaataggc gtatcacgag gccctttcgt cttcacctcg agaaatcata aaaaatttat
     3061 ttgctttgtg agcggataac aattataata gattcaattg tgagcggata acaatttcac
     3121 acagaattca ttaaagagga gaaattaacc atgggacatc accatcatca ccatcaccat
     3181 tcaaaaatcg aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt
     3241 ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat
     3301 ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt
     3361 atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc
     3421 accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac
     3481 aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa
     3541 gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg
     3601 aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg
     3661 ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa
     3721 gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt
     3781 aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa
     3841 ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa
     3901 gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt
     3961 ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc aaaagagttc
     4021 ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg
     4081 ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc
     4141 actatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc
     4201 tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa
     4261 gccctgaaag acgcgcagac taattcgagc tcgaacaaca acaacaataa caataacaac
     4321 aacctcggga tcgacgaaaa cctgtacctc acaagtttgt acaaaaaagc aggcttagag
     4381 aatctttatt ttcaggggat ggccgaaact atgaaagata ttacgatgaa gaacgatgaa
     4441 tcacaggaag aagaaatccc cgatcaattt ctctgctgcg tttgcctgga acttctttac
     4501 aagccaattg tgttatcttg tggtcatcta tcatgttttt ggtgcgtaca taagtccatg
     4561 aatggctttc gtgagtctca ttgtccgata tgtagagacc cgtatgttca ctttccctct
     4621 gtgtgccaga agctttattt tctgttaaag aagatgtacc cacttgctca taagaagaga
     4681 gaagaacaag ttttaaagga agagcaagaa cgagaatgtt tttctcctca gattgatctt
     4741 gttttggatt tgtctgtgtg tagtggagat tctctcaatg tctctgataa acagaaggtg
     4801 gaagagtgtt cgaatgcagc gaacttatta tctagttcat caagtagagg tgacattcca
     4861 tgtatcccca aaaatcaaga acccacagat gcaaaagctc ttaatgttca tgaaaatgaa
     4921 ttacttaagg ataacaaagt cagtaagcag atttcgaaag atgatttgct ctgttcagca
     4981 gctaaggagc tgcttgtacg acccgtagtt ctcaattgcg gacatgtgta ttgtgaagga
     5041 tgtgtagtag atatggctga agaaagcgaa aagatcaaat gtcaagagtg taatgtttgt
     5101 gacccaagag gatttccaaa agtttgtttg attcttgaac agcttttgga ggaaaacttt
     5161 cctgaagaat acaattcaag aagcagtaag gttcagaaaa cgctcgccca taatagcaaa
     5221 ggaaatattc aaagctatct caaagaaggc ccgtccttat caaacgacaa taacaatgat
     5281 gatccctggt tggcaaaccc tggatcaaat gttcactttg gagctggttg tgattcttgt
     5341 ggggtgtatc caatcatagg ggatcgatac agatgcaaag actgcaagga ggaaattggg
     5401 tatgaccttt gcaaagactg ttacgagact ccttcgaaag ttccagggag attcaaccag
     5461 caacacactc ctgaccacag gcttgagctt gcacggtctc ctcaggttct gatcaatttc
     5521 aattctatcg gtatccttct cggacccgtt atctcaaatg aaggcatgga tacagatgaa
     5581 ggcgaggaag ggcctcctgg ttcttctaat gagtcatcaa gcacagaatg atacccagct
     5641 ttcttgtaca aagtggtgat ggcgcgcctg taggacgtcg acggtaccat cgatacgcgt
     5701 tcgaagctta attagctgag cttggactcc tgttgataga tccagtaatg acctcagaac
     5761 tccatctgga tttgttcaga acgctcggtt gccgccgggc gttttttatt ggtgagaatc
     5821 caagctagct tggcgagatt ttcaggagct aaggaagcta aaatggagaa aaaaatcact
     5881 ggatatacca ccgttgatat atcccaatgg catcgtaaag aacattttga ggcatttcag
     5941 tcagttgctc aatgtaccta taaccagacc gttcagctgg atattacggc ctttttaaag
     6001 accgtaaaga aaaataagca caagttttat ccggccttta ttcacattct tgcccgcctg
     6061 atgaatgctc atccggaatt tcgtatggca atgaaagacg gtgagctggt gatatgggat
     6121 agtgttcacc cttgttacac cgttttccat gagcaaactg aaacgttttc atcgctctgg
     6181 agtgaatacc acgacgattt ccggcagttt ctacacatat attcgcaaga tgtggcgtgt
     6241 tacggtgaaa acctggccta tttccctaaa gggtttattg agaatatgtt tttcgtctca
     6301 gccaatccct gggtgagttt caccagtttt gatttaaacg tggccaatat ggacaacttc
     6361 ttcgcccccg ttttcaccat gggcaaatat tatacgcaag gcgacaaggt gctgatgccg
     6421 ctggcgattc aggttcatca tgccgtttgt gatggcttcc atgtcggcag aatgcttaat
     6481 gaattacaac agtactgcga tgagtggcag ggcggggcgt aattttttta aggcagttat
     6541 tggtgccctt aaacgcctgg ggtaatgact ctctagcttg aggcatcaaa taaaacgaaa
     6601 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct
     6661 gagtaggaca aatccgccct ctagattacg tgcagtcgat gataagctgt caaacatgag
     6721 aattgtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     6781 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     6841 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     6901 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     6961 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     7021 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
     7081 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     7141 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     7201 ccgttccgct atcggctgaa tttgattgcg ag
//
