LOCUS       sequence-450906-        6205 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GGG-TitinV15P-43-K-8-C-25-intein-CBD.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6205)
  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 6205)
  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: pAM95
FEATURES             Location/Qualifiers
     source          1..6205
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     22..39
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    54..193
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(119..138)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     279..301
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(295..486)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1295..2377)
                     /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     2339..2358
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2378..2455)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2501..2520
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2501..2519
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2520..2544
                     /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             3101..3694
                     /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             3725..3880
                     /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(3903..3921)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3917..3964
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4001..4456
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4088..4107)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4298..4319
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4548..5363)
                     /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(4548..5363)
                     /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     5271..5290
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5355..5374
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5485..6073
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5974..5993
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
       61 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
      121 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
      181 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
      241 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
      301 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
      361 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
      421 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
      481 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
      541 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
      601 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
      661 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
      721 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
      781 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
      841 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
      901 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
      961 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     1021 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     1081 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     1141 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     1201 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     1261 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     1321 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     1381 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     1441 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     1501 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     1561 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     1621 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     1681 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     1741 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     1801 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     1861 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     1921 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     1981 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     2041 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     2101 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     2161 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     2221 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     2281 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     2341 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     2401 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     2461 tctcgacgct ctcccttatg cgactcctgc attaggaaat taatacgact cactataggg
     2521 gaattgtgag cggataacaa ttcccctgta gaaataattt tgtttaactt taataaggag
     2581 atataccatg ggtggcggac taatagaagt ggaacgccct ctgtacggag tagaggtgtt
     2641 tccgggtgaa acagcccact ttgaaattga actttctgaa cctgatgttc acggccagtg
     2701 gcgcctgcgc ggacagcctt tggcagcttc ccctgactgt gaaatcattg aggatggacg
     2761 ccgccatatt ctgatccttc ataactgtca gctgggtatg acaggagagg tttccttcca
     2821 ggctgctaat acccgctctg cagccaatct gcgcgtgcgc gaattgggtg gagcaggacc
     2881 gccaccatat agtgcagcaa acgacgaaaa ctacgcttta gcagcgcatg gtggcgctca
     2941 tacctttaat aacgagaatg ttagcgcaca cacattcaac aatgaaaatg tcagtaaaca
     3001 tacctttaac aatgaaaacg tgtgtaggct cgggggagcg gctagcattg ctgtgcaagc
     3061 gccagcacaa cataccttca acaatgaaaa cgtttcctat tgcatcacgg gagatgcact
     3121 agttgcccta cccgagggcg agtcggtacg catcgccgac atcgtgccgg gtgcgcggcc
     3181 caacagtgac aacgccatcg acctgaaagt ccttgaccgg catggcaatc ccgtgctcgc
     3241 cgaccggctg ttccactccg gcgagcatcc ggtgtacacg gtgcgtacgg tcgaaggtct
     3301 gcgtgtgacg ggcaccgcga accacccgtt gttgtgtttg gtcgacgtcg ccggggtgcc
     3361 gaccctgctg tggaagctga tcgacgaaat caagccgggc gattacgcgg tgattcaacg
     3421 cagcgcattc agcgtcgact gtgcaggttt tgcccgcggg aaacccgaat ttgcgcccac
     3481 aacctacaca gtcggcgtcc ctggactggt gcgtttcttg gaagcacacc accgagaccc
     3541 ggacgcccaa gctatcgccg acgagctgac cgacgggcgg ttctactacg cgaaagtcgc
     3601 cagtgtcacc gacgccggcg tgcagccggt gtatagcctt cgtgtcgaca cggcagacca
     3661 cgcgtttatc acgaacgggt tcgtcagcca cgctactggc ctcaccggtc tgaactcagg
     3721 cctcacgaca aatcctggtg tatccgcttg gcaggtcaac acagcttata ctgcgggaca
     3781 attggtcaca tataacggca agacgtataa atgtttgcag ccccacacct ccttggcagg
     3841 atgggaacca tccaacgttc ctgccttgtg gcagcttcaa taattaacct aggctgctgc
     3901 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     3961 tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca
     4021 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     4081 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     4141 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     4201 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     4261 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     4321 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     4381 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     4441 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     4501 tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca
     4561 tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga
     4621 aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga
     4681 tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc
     4741 tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag
     4801 aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg
     4861 tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga
     4921 cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc
     4981 aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc
     5041 tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg
     5101 ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc
     5161 tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca
     5221 tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc
     5281 catttatacc catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac
     5341 gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt
     5401 tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     5461 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     5521 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     5581 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     5641 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     5701 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     5761 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     5821 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     5881 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     5941 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     6001 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     6061 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     6121 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     6181 ctttgagtga gctgataccg ctcgc
//
