LOCUS       sequence-450911-        6136 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GGG-TitinV15P-43-K-11-intein-CBD.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6136)
  AUTHORS   Bard JAM, Bashore C, Dong KC, Martin A
  TITLE     The 26S Proteasome Utilizes a Kinetic Gateway to Prioritize 
            Substrate Degradation.
  JOURNAL   Cell. 2019 Apr 4;177(2):286-298.e15. doi: 
            10.1016/j.cell.2019.02.031. Epub 2019 Mar 28.
  PUBMED    30929903
REFERENCE   2  (bases 1 to 6136)
  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: 
            "10.1016/j.cell.2019.02.031"; journalName: "Cell"; date: "2019-04-4-
            4"; volume: "177"; issue: "2"; pages: "286-298.e15"
COMMENT     Sequence Label: pAM104
FEATURES             Location/Qualifiers
     source          1..6136
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     104..123
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        104..122
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    123..147
                     /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             635..1228
                     /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             1259..1414
                     /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(1437..1455)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1451..1498
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1535..1990
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1622..1641)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1832..1853
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2082..2897)
                     /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(2082..2897)
                     /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     2805..2824
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2889..2908
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3019..3607
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3508..3527
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3761..3778
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3793..3932
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3858..3877)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4018..4040
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4034..4225)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5034..6116)
                     /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     6078..6097
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(join(6117..6136,1..58))
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg
       61 ggatctcgac gctctccctt atgcgactcc tgcattagga aattaatacg actcactata
      121 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
      181 gagatatacc atgggtggcg gactaataga agtggaacgc cctctgtacg gagtagaggt
      241 gtttccgggt gaaacagccc actttgaaat tgaactttct gaacctgatg ttcacggcca
      301 gtggcgcctg cgcggacagc ctttggcagc ttcccctgac tgtgaaatca ttgaggatgg
      361 acgccgccat attctgatcc ttcataactg tcagctgggt atgacaggag aggtttcctt
      421 ccaggctgct aatacccgct ctgcagccaa tctgcgcgtg cgcgaattgg gtggagcagg
      481 accgccacca tatagtgcag caaacgacga aaactacgct ttagcagcgc atggtggcaa
      541 acataccttt aataacgaga atgttagcgc acacacattc aacaatgaaa atgtcagtaa
      601 acataccttt aacaatgaaa acgtgagttg ttattgcatc acgggagatg cactagttgc
      661 cctacccgag ggcgagtcgg tacgcatcgc cgacatcgtg ccgggtgcgc ggcccaacag
      721 tgacaacgcc atcgacctga aagtccttga ccggcatggc aatcccgtgc tcgccgaccg
      781 gctgttccac tccggcgagc atccggtgta cacggtgcgt acggtcgaag gtctgcgtgt
      841 gacgggcacc gcgaaccacc cgttgttgtg tttggtcgac gtcgccgggg tgccgaccct
      901 gctgtggaag ctgatcgacg aaatcaagcc gggcgattac gcggtgattc aacgcagcgc
      961 attcagcgtc gactgtgcag gttttgcccg cgggaaaccc gaatttgcgc ccacaaccta
     1021 cacagtcggc gtccctggac tggtgcgttt cttggaagca caccaccgag acccggacgc
     1081 ccaagctatc gccgacgagc tgaccgacgg gcggttctac tacgcgaaag tcgccagtgt
     1141 caccgacgcc ggcgtgcagc cggtgtatag ccttcgtgtc gacacggcag accacgcgtt
     1201 tatcacgaac gggttcgtca gccacgctac tggcctcacc ggtctgaact caggcctcac
     1261 gacaaatcct ggtgtatccg cttggcaggt caacacagct tatactgcgg gacaattggt
     1321 cacatataac ggcaagacgt ataaatgttt gcagccccac acctccttgg caggatggga
     1381 accatccaac gttcctgcct tgtggcagct tcaataatta acctaggctg ctgccaccgc
     1441 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     1501 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     1561 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     1621 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     1681 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     1741 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     1801 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     1861 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     1921 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     1981 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     2041 tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc
     2101 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
     2161 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
     2221 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
     2281 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
     2341 aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
     2401 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat
     2461 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     2521 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     2581 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     2641 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     2701 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     2761 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     2821 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc
     2881 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
     2941 gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     3001 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     3061 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     3121 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     3181 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     3241 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     3301 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     3361 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     3421 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     3481 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     3541 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     3601 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     3661 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     3721 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     3781 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     3841 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     3901 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     3961 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     4021 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     4081 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     4141 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     4201 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     4261 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     4321 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     4381 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     4441 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     4501 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     4561 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     4621 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     4681 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     4741 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     4801 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     4861 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     4921 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     4981 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     5041 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     5101 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     5161 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     5221 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     5281 ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac
     5341 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     5401 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     5461 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     5521 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     5581 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     5641 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     5701 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     5761 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     5821 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     5881 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     5941 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     6001 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     6061 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     6121 accaccctga attgac
//
