LOCUS       sequence-449621-        6147 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Titin (V15P) substrate for 26S proteasome.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6147)
  AUTHORS   Jonsson E, Htet ZM, Bard JAM, Dong KC, Martin A
  TITLE     Ubiquitin modulates 26S proteasome conformational dynamics and 
            promotes substrate degradation.
  JOURNAL   Sci Adv. 2022 Dec 23;8(51):eadd9520. doi: 10.1126/sciadv.add9520. 
            Epub 2022 Dec 23.
  PUBMED    36563145
REFERENCE   2  (bases 1 to 6147)
  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: "Sci Adv."; 
            date: "2022-12-23"; volume: "8(51):eadd9520. doi"; pages: " 
            10.1126/sciadv.add9520. Epub 2022 Dec 23"
COMMENT     Sequence Label: pAM355 pET28a-GGG-Titin[V15P]-23-K-24-C-10
FEATURES             Location/Qualifiers
     source          1..6147
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     26..45
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     279..296
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    311..450
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(376..395)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     536..558
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(552..743)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1552..2634)
                     /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"
     primer_bind     2596..2615
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2635..2712)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2758..2777
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2758..2776
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2777..2801
                     /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)."
     CDS             3301..3894
                     /codon_start=1
                     /gene="Mycobacterium xenopi gyrA"
                     /product="modified GyrA intein (Southworth et al., 1999)"
                     /label=Mxe GyrA intein
                     /note="N-terminal cleavage of this intein can be induced by
                     thiols."
                     /translation="CITGDALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPV
                     LADRLFHSGEHPVYTVRTVEGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAV
                     IQRSAFSVDCAGFARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYY
                     AKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHA"
     CDS             3925..4080
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
                     PALWQLQ"
     primer_bind     complement(4103..4121)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4117..4164
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4201..4656
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4288..4307)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4498..4519
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4748..5563)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(4748..5563)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5471..5490
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5555..5574
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(5685..6147,1..125)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
       61 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
      121 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
      181 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
      241 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
      301 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
      361 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
      421 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
      481 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
      541 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
      601 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
      661 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
      721 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
      781 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
      841 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
      901 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
      961 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     1021 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     1081 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     1141 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     1201 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     1261 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     1321 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     1381 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     1441 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     1501 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     1561 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     1621 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     1681 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     1741 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     1801 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
     1861 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     1921 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     1981 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     2041 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     2101 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     2161 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     2221 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     2281 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     2341 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     2401 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     2461 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     2521 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     2581 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     2641 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     2701 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaaattaa
     2761 tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctgtagaa ataattttgt
     2821 ttaactttaa taaggagata taccatgggt ggcggactaa tagaagtgga acgccctctg
     2881 tacggagtag aggtgtttcc gggtgaaaca gcccactttg aaattgaact ttctgaacct
     2941 gatgttcacg gccagtggcg cctgcgcgga cagcctttgg cagcttcccc tgactgtgaa
     3001 atcattgagg atggacgccg ccatattctg atccttcata actgtcagct gggtatgaca
     3061 ggagaggttt ccttccaggc tgctaatacc cgctctgcag ccaatctgcg cgtgcgcgaa
     3121 ttgggtggag caggaccgcc accatatagt gcagcaaacg acgaaaacta cgctttagca
     3181 gcgcatggtg gcaaacacac attcaacaat gaaaatgtca gtgcaaggct cgggggagcg
     3241 gctagcattg ctgtgcaagc gccagcatgt cataccttca acaatgaaaa cgtttcctat
     3301 tgcatcacgg gagatgcact agttgcccta cccgagggcg agtcggtacg catcgccgac
     3361 atcgtgccgg gtgcgcggcc caacagtgac aacgccatcg acctgaaagt ccttgaccgg
     3421 catggcaatc ccgtgctcgc cgaccggctg ttccactccg gcgagcatcc ggtgtacacg
     3481 gtgcgtacgg tcgaaggtct gcgtgtgacg ggcaccgcga accacccgtt gttgtgtttg
     3541 gtcgacgtcg ccggggtgcc gaccctgctg tggaagctga tcgacgaaat caagccgggc
     3601 gattacgcgg tgattcaacg cagcgcattc agcgtcgact gtgcaggttt tgcccgcggg
     3661 aaacccgaat ttgcgcccac aacctacaca gtcggcgtcc ctggactggt gcgtttcttg
     3721 gaagcacacc accgagaccc ggacgcccaa gctatcgccg acgagctgac cgacgggcgg
     3781 ttctactacg cgaaagtcgc cagtgtcacc gacgccggcg tgcagccggt gtatagcctt
     3841 cgtgtcgaca cggcagacca cgcgtttatc acgaacgggt tcgtcagcca cgctactggc
     3901 ctcaccggtc tgaactcagg cctcacgaca aatcctggtg tatccgcttg gcaggtcaac
     3961 acagcttata ctgcgggaca attggtcaca tataacggca agacgtataa atgtttgcag
     4021 ccccacacct ccttggcagg atgggaacca tccaacgttc ctgccttgtg gcagcttcaa
     4081 taattaacct aggctgctgc caccgctgag caataactag cataacccct tggggcctct
     4141 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
     4201 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     4261 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     4321 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     4381 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     4441 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     4501 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     4561 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     4621 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
     4681 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa
     4741 ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat
     4801 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc
     4861 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa
     4921 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag
     4981 tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa
     5041 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc
     5101 gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag
     5161 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat
     5221 caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc
     5281 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca
     5341 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt
     5401 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt
     5461 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta
     5521 atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac
     5581 tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg
     5641 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     5701 ctgcgcgtaa tctgctgctt gcaaacaaaa aaccaccgct accagcggtg gtttgtttgc
     5761 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     5821 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     5881 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     5941 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     6001 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     6061 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     6121 atccggtaag cggcagggtc ggaacag
//
