LOCUS       sequence-455225-        7453 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pRS305 3XFLAG-HRV3C-Rpn5 WT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7453)
  AUTHORS   Greene ER, Goodall EA, de la Pena AH, Matyskiela ME, Lander GC, 
            Martin A
  TITLE     Specific lid-base contacts in the 26s proteasome control the 
            conformational switching required for substrate degradation.
  JOURNAL   Elife. 2019 Nov 28;8:e49806. doi: 10.7554/eLife.49806.
  PUBMED    31778111
REFERENCE   2  (bases 1 to 7453)
  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: "Elife."; 
            date: "2019-11-28"; volume: "8:e49806. doi"; pages: " 
            10.7554/eLife.49806"
COMMENT     Sequence Label: pAM200
FEATURES             Location/Qualifiers
     source          1..7453
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(17..424)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     rep_origin      complement(725..1180)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(862..883)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1074..1093
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1306..1328
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1320..1337
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1321..1337
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1344..1363
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1344..1362
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(2817..2840)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(2850..2915)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     promoter        complement(3451..3469)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3490..3506)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3490..3506)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3503..3525)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3514..3530
                     /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)."
     promoter        complement(join(3538..3544,3545..3562,3563..3568))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3583..3604
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3721..3738)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3892..4480)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3972..3991)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4651..5442,5443..5511))
                     /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     5274..5293
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5512..5616)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5684..5702
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5740..5762)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5862..5881
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(join(6375..7453,1..16))
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
ORIGIN
        1 tcttaggggc agacattaga atggtatatc cttgaaatat atatatatat attgctgaaa
       61 tgtaaaaggt aagaaaagtt agaaagtaag acgattgcta accacctatt ggaaaaaaca
      121 ataggtcctt aaataatatt gtcaacttca agtattgtga tgcaagcatt tagtcatgaa
      181 cgcttctcta ttctatatga aaagccggtt ccggcgctct cacctttcct ttttctccca
      241 atttttcagt tgaaaaaggt atatgcgtca ggcgacctct gaaattaaca aaaaatttcc
      301 agtcatcgaa tttgattctg tgcgatagcg cccctgtgtg ttctcgttat gttgaggaaa
      361 aaaataatgg ttgctaagag attcgaactc ttgcatctta cgatacctga gtattcccac
      421 agttaactgc ggtcaagata tttcttgaat caggcgcctt agaccgctcg gccaaacaac
      481 caattacttg ttgagaaata gagtataatt atcctataaa tataacgttt ttgaacacac
      541 atgaacaagg aagtacagga caattgattt tgaagagaat gtggattttg atgtaattgt
      601 tgggattcca tttttaataa ggcaataata ttaggtatgt agatatacta gaagttctcc
      661 tcgaccggtc gatatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat
      721 caggaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
      781 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
      841 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
      901 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
      961 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     1021 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     1081 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     1141 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt ccattcgcca ttcaggctgc
     1201 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag
     1261 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt
     1321 gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaattg gagctcataa
     1381 ttttatctca cgtacgtaaa tacacatttc gattagtgac agatttaaaa tcaaaaccgg
     1441 atctgagata atccgacagt tactcgaaaa tctcttcatt tagcttgcag accgtgcatg
     1501 atttcctctt ttgtaattaa atggcctatt gtttctatat gttctaatag ttcgtcaaca
     1561 ttatgtgacc attcgttcaa taattggctt gagttttttg gtttttcaaa attcacgatt
     1621 ttggctgggc gattaacttt agcgtatatg atgccctggt ttaccaaatc actgatgtat
     1681 gtttccgtct ggctctccgt taggtccagc aattcattca atcttagtaa agtaattctg
     1741 gaatagtatt cggaaatgac tcttaaattg tgctcgatga cccttttttg taaatcttcc
     1801 cagtgatgct tattagcttc tccaccaaat gccaaatcat cctcatttaa gacgggctca
     1861 taggtttttt gaacaattgg ccatctcatc aactcattcg tagtaaacaa ttttactaaa
     1921 gattcttggc tttctaattt tttcaggttg ttatcattct ggattttgtg aattaaatca
     1981 ttttgtaaat tgccgtaagg tgaaaggact aagaaatata caatgtgcga taaaacaggt
     2041 ttccacttag cctcatctga tttaatggcg tctgtttgat aaatttcttg cagatactgc
     2101 gcaacttcta ggtattctct cttgtgcaaa ctaattttta ccagaagatt ataatattct
     2161 agcttcaatg actcgtattt tggattttta aaagtttttt tcagaatttt tcttgaaagc
     2221 accgtggctt gggaataatc accttttaat atactcaatt ccatttgctc taatataaac
     2281 tgaattttct cagacatttc catggagcca taggtctcaa cctgtaactc acacaagatg
     2341 tctgcagctt catcaatctt accctcttct ttcttaattt ctaccaaatc tttggtgacc
     2401 ctagctcttt ccacttctac aaatattttg ttctctgtaa ccaccctgat agtttcaatg
     2461 acactaattc tggtgtttaa atccaaagat ttcgagcttt tcaaatattc cataaccttt
     2521 tgtatcatat actgaattga caatttcaac tgaccatgct tttttgagag tagagtcaat
     2581 tgctcattta ggtcgtccca cttattcctt gatgctagca gatctacaat cttggccaaa
     2641 acttctttcg aggaggccag atctgaagct tgtctggttt tcttctccaa cactaacagt
     2701 tggtctaaag cagagttaca atcattttga gcgagcgaat cgatcttagg aaactcttcc
     2761 ttcaaaattt ggctataatc cttgtcagcc ttaattggtg catctcttga catacctgga
     2821 ccttgaaata aaacttccaa accgcttcct ttatcatcat catctttgta atcaatatca
     2881 tgatctttgt aatcgccatc atgatctttg taatccatct ttctctacgt ttactctagc
     2941 aacaaaaatg gtactgagcc cagcagtata ttcctctact ctcttaattc aagattatct
     3001 ctgatgtagt gtcctgttat ggtttttcct tagattcccc tttgcatttt gccaccgaaa
     3061 agtcaaaaag atcacataca agacgggtaa tattggggcg atgagccgta cagaacgagt
     3121 gaaaaatttt aggatttatt aggaataata atgataattt agagatgagt atttagcgag
     3181 aagcatctga aggttgcact aattattact gtaaatagta cagttgagaa gcaaatacaa
     3241 gggaagatca aatacatggc cggttcacaa cttaagaatt taaaggccgc cttgaaggcc
     3301 cgtggactca caggtcaaac aaacgtcaaa agtaagaata aaaaaaattc caaaagacaa
     3361 gcgaaggagt atgatcgtga agaaaaaaaa aaggccattg ctgaaatcag agaagagtct
     3421 cgaggggggg cccggtacca gcttttgttc cctttagtga gggttaattt cgagcttggc
     3481 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     3541 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     3601 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     3661 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     3721 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     3781 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     3841 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     3901 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     3961 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     4021 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     4081 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     4141 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     4201 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     4261 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     4321 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     4381 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     4441 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     4501 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     4561 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     4621 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     4681 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     4741 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     4801 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     4861 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     4921 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     4981 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     5041 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     5101 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     5161 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     5221 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     5281 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     5341 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     5401 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     5461 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     5521 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     5581 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     5641 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag
     5701 gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     5761 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     5821 gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt
     5881 actgagagtg caccatatcg actacgtcgt taaggccgtt tctgacagag taaaattctt
     5941 gagggaactt tcaccattat gggaaatggt tcaagaaggt attgacttaa actccatcaa
     6001 atggtcaggt cattgagtgt tttttatttg ttgtattttt ttttttttag agaaaatcct
     6061 ccaatatata aattaggaat catagtttca tgattttctg ttacacctaa ctttttgtgt
     6121 ggtgccctcc tccttgtcaa tattaatgtt aaagtgcaat tctttttcct tatcacgttg
     6181 agccattagt atcaatttgc ttacctgtat tcctttacat cctccttttt ctccttcttg
     6241 ataaatgtat gtagattgcg tatatagttt cgtctaccct atgaacatat tccattttgt
     6301 aatttcgtgt cgtttctatt atgaatttca tttataaagt ttatgtacaa atatcataaa
     6361 aaaagagaat ctttttaagc aaggattttc ttaacttctt cggcgacagc atcaccgact
     6421 tcggtggtac tgttggaacc acctaaatca ccagttctga tacctgcatc caaaaccttt
     6481 ttaactgcat cttcaatggc cttaccttct tcaggcaagt tcaatgacaa tttcaacatc
     6541 attgcagcag acaagatagt ggcgataggg ttgaccttat tctttggcaa atctggagca
     6601 gaaccgtggc atggttcgta caaaccaaat gcggtgttct tgtctggcaa agaggccaag
     6661 gacgcagatg gcaacaaacc caaggaacct gggataacgg aggcttcatc ggagatgata
     6721 tcaccaaaca tgttgctggt gattataata ccatttaggt gggttgggtt cttaactagg
     6781 atcatggcgg cagaatcaat caattgatgt tgaaccttca atgtagggaa ttcgttcttg
     6841 atggtttcct ccacagtttt tctccataat cttgaagagg ccaaaacatt agctttatcc
     6901 aaggaccaaa taggcaatgg tggctcatgt tgtagggcca tgaaagcggc cattcttgtg
     6961 attctttgca cttctggaac ggtgtattgt tcactatccc aagcgacacc atcaccatcg
     7021 tcttcctttc tcttaccaaa gtaaatacct cccactaatt ctctgacaac aacgaagtca
     7081 gtacctttag caaattgtgg cttgattgga gataagtcta aaagagagtc ggatgcaaag
     7141 ttacatggtc ttaagttggc gtacaattga agttctttac ggatttttag taaaccttgt
     7201 tcaggtctaa cactaccggt accccattta ggaccaccca cagcacctaa caaaacggca
     7261 tcagccttct tggaggcttc cagcgcctca tctggaagtg gaacacctgt agcatcgata
     7321 gcagcaccac caattaaatg attttcgaaa tcgaacttga cattggaacg aacatcagaa
     7381 atagctttaa gaaccttaat ggcttcggct gtgatttctt gaccaacgtg gtcacctggc
     7441 aaaacgacga tct
//
