LOCUS       sequence-450054-        6244 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of GFP-titin-ssrA substrate for 26S proteasome.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6244)
  AUTHORS   Bashore C, Dambacher CM, Goodall EA, Matyskiela ME, Lander GC, 
            Martin A
  TITLE     Ubp6 deubiquitinase controls conformational dynamics and substrate 
            degradation of the 26S proteasome.
  JOURNAL   Nat Struct Mol Biol. 2015 Sep;22(9):712-9. doi: 10.1038/nsmb.3075. 
            Epub 2015 Aug 24.
  PUBMED    26301997
REFERENCE   2  (bases 1 to 6244)
  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"; doi: "10.1038/nsmb.3075";
            journalName: "Nat Struct Mol Biol"; date: "2015-09"; volume: "22"; 
            issue: "9"; pages: "712-9"
COMMENT     Sequence Label: pAM420 GFP-titin-ssrA substrate
FEATURES             Location/Qualifiers
     source          1..6244
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     605..624
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(644..721)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     927..946
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1034..1053
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1034..1052
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1053..1077
                     /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             1092..1114
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2285..2302
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2325..2343)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2339..2386
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2423..2878
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2510..2529)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2720..2741
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2993..3784,3785..3853))
                     /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     3616..3635
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3854..3946)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4027..4615
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4516..4535
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4769..4786
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4801..4940
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4866..4885)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5026..5048
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5042..5233)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5805..6244,1..643))
                     /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 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
       61 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
      121 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
      181 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
      241 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
      301 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
      361 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
      421 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
      481 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
      541 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
      601 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
      661 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
      721 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
      781 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
      841 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
      901 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
      961 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatcgatctc
     1021 gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa caattcccct
     1081 ctagaaataa ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcactg
     1141 ccgcggcgag gagctgttca ccggcgtggt gcccatcctg gtggagctgg acggcgacgt
     1201 gaacggccac cgcttcagcg tgcgcggcga gggcgagggc gacgccacca acggccgcct
     1261 gaccctgcgc ttcatctgca ccaccggccg cctgcccgtg ccctggccca ccctggtgac
     1321 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatgc gccgccacga
     1381 cttcttccgc agcgccatgc ccgagggcta cgtgcaggag cgcaccatca gcttccgcga
     1441 cgacggcacc taccgcaccc gcgccgaggt gcgcttcgag ggcgacaccc tggtgaaccg
     1501 catcgagctg cgcggcatcg acttccgcga ggacggcaac atcctgggcc accgcctgga
     1561 gtacaacttc aacagccaca acgtgtacat caccgccgac cgccagcgca acggcatccg
     1621 cgccaacttc cgcatccgcc acaacgtgga ggacggcagc gtgcagctgg ccgaccacta
     1681 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     1741 cacccagagc gtgctgagcc gcgaccccaa cgagcgccgc gaccacatgg tgctgctgga
     1801 gttcgtgacc gccgccggca tcacccacgg catggacgag ctgtaccgcg ccatgggcag
     1861 cagccatcac tgcctaatag aagtggaacg ccctctgtac ggagtagagg tgtttccggg
     1921 tgaaacagcc cactttgaaa ttgaactttc tgaacctgat gttcacggcc agtggcgcct
     1981 gcgcggacag cctttggcag cttcccctga ctgtgaaatc attgaggatg gacgccgcca
     2041 tattctgatc cttcataact gtcagctggg tatgacagga gaggtttcct tccaggctgc
     2101 taatacccgc tctgcagcca atctgcgcgt gcgcgaattg ggtaccctcg aggcagcaaa
     2161 cgacgaaaac tacgctttag cagcgcatgg tggcaaacac acattcaaca atgaaaatgt
     2221 cagtgcaagg ctcgggggag cgagcattgc tgtgcaagcg ccagcacaac cgccaccata
     2281 tagtcaccat caccatcacc attaattaac ctaggctgct gccaccgctg agcaataact
     2341 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
     2401 tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     2461 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     2521 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     2581 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     2641 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     2701 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     2761 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     2821 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttct
     2881 ggcggcacga tggcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     2941 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     3001 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     3061 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     3121 caatgatacc gcgagaacca cgctcaccgg ctccagattt atcagcaata aaccagccag
     3181 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     3241 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     3301 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     3361 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     3421 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     3481 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     3541 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     3601 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     3661 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     3721 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     3781 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     3841 gttgaatact catactcttc ctttttcaat catgattgaa gcatttatca gggttattgt
     3901 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg tcatgaccaa
     3961 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     4021 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     4081 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     4141 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     4201 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     4261 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     4321 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     4381 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     4441 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     4501 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     4561 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     4621 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     4681 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     4741 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     4801 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     4861 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     4921 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     4981 cttgtctgct cccggcatcc gcttacagac aagctgtgac agtctccggg agctgcatgt
     5041 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     5101 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
     5161 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     5221 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     5281 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     5341 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     5401 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     5461 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     5521 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
     5581 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
     5641 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg ctagtcatgc
     5701 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     5761 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     5821 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     5881 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgaaacggg caacagctga
     5941 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     6001 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     6061 gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg
     6121 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     6181 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     6241 cgtt
//
