LOCUS       sequence-175083-        7026 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 7026)
  TITLE     PRT1 clones
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7026)
  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            "Unpublished"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-C29A
FEATURES             Location/Qualifiers
     source          1..7026
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     701..720
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(740..817)
                     /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(901..923)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1009..1149
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1064..1083
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1164..1181)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1335..1923)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1415..1434)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2094..2885,2886..2954))
                     /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     2717..2736
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2955..3059)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3127..3145
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(3169..3183,3184..3189,3190..3206,3207..3213)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    3189..3205
                     /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    3221..3237
                     /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)."
     primer_bind     3226..3248
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4172..4195
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    4268..4292
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4295..4315
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     protein_bind    complement(5550..5574)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      5642..5736
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             5780..6439
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(5846..5865)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      6504..6590
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(6683..7026,1..739))
                     /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 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
       61 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
      121 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
      181 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
      241 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
      301 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
      361 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
      421 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
      481 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
      541 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
      601 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
      661 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
      721 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
      781 ccataccgcg aaaggttttg caccattcga tggtgtcgga atttcgggca gcgttgggtc
      841 ctggccacgg gtgcgcatga tctagagctg cctcgcgcgt ttcggtgatg acggtgaaaa
      901 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
      961 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac
     1021 ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt
     1081 gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     1141 cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     1201 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     1261 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     1321 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     1381 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     1441 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     1501 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     1561 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     1621 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     1681 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     1741 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     1801 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     1861 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     1921 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     1981 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     2041 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     2101 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     2161 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     2221 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     2281 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     2341 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     2401 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     2461 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     2521 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     2581 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     2641 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     2701 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     2761 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     2821 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     2881 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     2941 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     3001 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     3061 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
     3121 tataaaaata ggcgtatcac gaggcccttt cgtcttcacc tcgagaaatc ataaaaaatt
     3181 tatttgcttt gtgagcggat aacaattata atagattcaa ttgtgagcgg ataacaattt
     3241 cacacaggtt gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt
     3301 tggtggctac gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga
     3361 caagctgtat ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc
     3421 gatcgctgtt gaagcgttat cgctgattta taacaaagat ctgctgccga acccgccaaa
     3481 aacctgggaa gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct
     3541 gatgttcaac ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta
     3601 tgcgttcaag tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg
     3661 cgcgaaagcg ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga
     3721 caccgattac tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa
     3781 cggcccgtgg gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact
     3841 gccgaccttc aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa
     3901 cgccgccagt ccgaacaaag agctggcaaa agagttcctc gaaaactatc tgctgactga
     3961 tgaaggtctg gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta
     4021 cgaggaagag ttggcgaaag atccacgtat tgccgccact atggaaaacg cccagaaagg
     4081 tgaaatcatg ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt
     4141 gatcaacgcc gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg cgcagactaa
     4201 ttcgagctcg aacaacaaca acaataacaa taacaacaac ctcgggatcg acgaaaacct
     4261 gtacctcaca agtttgtaca aaaaagcagg cttagagaat ctttattttc aggggatggc
     4321 cgaaactatg aaagatatta cgatgaagaa cgatgaatca caggaagaag aaatccccga
     4381 tcaatttctc tgctgcgttg ctctggaact tctttacaag ccaattgtgt tatcttgtgg
     4441 tcatctatca tgtttttggt gcgtacataa gtccatgaat ggctttcgtg agtctcattg
     4501 tccgatatgt agagacccgt atgttcactt tccctctgtg tgccagaagc tttattttct
     4561 gttaaagaag atgtacccac ttgctcataa gaagagagaa gaacaagttt taaaggaaga
     4621 gcaagaacga gaatgttttt ctcctcagat tgatcttgtt ttggatttgt ctgtgtgtag
     4681 tggagattct ctcaatgtct ctgataaaca gaaggtggaa gagtgttcga atgcagcgaa
     4741 cttattatct agttcatcaa gtagaggtga cattccatgt atccccaaaa atcaagaacc
     4801 cacagatgca aaagctctta atgttcatga aaatgaatta cttaaggata acaaagtcag
     4861 taagcagatt tcgaaagatg atttgctctg ttcagcatgt aaggagctgc ttgtacgacc
     4921 cgtagttctc aattgcggac atgtgtattg tgaaggatgt gtagtagata tggctgaaga
     4981 aagcgaaaag atcaaatgtc aagagtgtaa tgtttgtgac ccaagaggat ttccaaaagt
     5041 ttgtttgatt cttgaacagc ttttggagga aaactttcct gaagaataca attcaagaag
     5101 cagtaaggtt cagaaaacgc tcgcccataa tagcaaagga aatattcaaa gctatctcaa
     5161 agaaggcccg tccttatcaa acgacaataa caatgatgat ccctggttgg caaaccctgg
     5221 atcaaatgtt cactttggag ctggttgtga ttcttgtggg gtgtatccaa tcatagggga
     5281 tcgatacaga tgcaaagact gcaaggagga aattgggtat gacctttgca aagactgtta
     5341 cgagactcct tcgaaagttc cagggagatt caaccagcaa cacactcctg accacaggct
     5401 tgagcttgca cggtctcctc aggttctgat caatttcaat tctatcggta tccttctcgg
     5461 acccgttatc tcaaatgaag gcatggatac agatgaaggc gaggaagggc ctcctggttc
     5521 ttctaatgag tcatcaagca cagaatgata cccagctttc ttgtacaaag tggtgatggc
     5581 gcgcctgtag gacgtcgacg gtaccatcga tacgcgttcg aagcttaatt agctgagctt
     5641 ggactcctgt tgatagatcc agtaatgacc tcagaactcc atctggattt gttcagaacg
     5701 ctcggttgcc gccgggcgtt ttttattggt gagaatccaa gctagcttgg cgagattttc
     5761 aggagctaag gaagctaaaa tggagaaaaa aatcactgga tataccaccg ttgatatatc
     5821 ccaatggcat cgtaaagaac attttgaggc atttcagtca gttgctcaat gtacctataa
     5881 ccagaccgtt cagctggata ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa
     5941 gttttatccg gcctttattc acattcttgc ccgcctgatg aatgctcatc cggaatttcg
     6001 tatggcaatg aaagacggtg agctggtgat atgggatagt gttcaccctt gttacaccgt
     6061 tttccatgag caaactgaaa cgttttcatc gctctggagt gaataccacg acgatttccg
     6121 gcagtttcta cacatatatt cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt
     6181 ccctaaaggg tttattgaga atatgttttt cgtctcagcc aatccctggg tgagtttcac
     6241 cagttttgat ttaaacgtgg ccaatatgga caacttcttc gcccccgttt tcaccatggg
     6301 caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgattcagg ttcatcatgc
     6361 cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa ttacaacagt actgcgatga
     6421 gtggcagggc ggggcgtaat ttttttaagg cagttattgg tgcccttaaa cgcctggggt
     6481 aatgactctc tagcttgagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct
     6541 ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccctcta
     6601 gattacgtgc agtcgatgat aagctgtcaa acatgagaat tgtgcctaat gagtgagcta
     6661 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     6721 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     6781 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     6841 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     6901 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     6961 agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     7021 tctgat
//
