LOCUS       sequence-450907-        6136 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GGG-TitinV15P-23-K-19-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: pAM105
FEATURES             Location/Qualifiers
     source          1..6136
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     145..167
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(161..352)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1161..2243)
                     /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     2205..2224
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2244..2321)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2367..2386
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2367..2385
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2386..2410
                     /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             2898..3491
                     /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             3522..3677
                     /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(3700..3718)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3714..3761
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3798..4253
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3885..3904)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4095..4116
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4345..5160)
                     /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(4345..5160)
                     /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     5068..5087
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5152..5171
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5282..5870
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5771..5790
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6024..6041
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(6056..6136,1..59)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(join(6121..6136,1..4))
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
       61 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
      121 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
      181 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
      241 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
      301 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
      361 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
      421 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
      481 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
      541 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
      601 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
      661 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
      721 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
      781 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa
      841 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
      901 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
      961 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     1021 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     1081 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     1141 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     1201 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     1261 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     1321 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     1381 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     1441 atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     1501 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     1561 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     1621 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     1681 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     1741 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     1801 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     1861 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     1921 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     1981 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     2041 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     2101 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     2161 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     2221 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     2281 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     2341 cttatgcgac tcctgcatta ggaaattaat acgactcact ataggggaat tgtgagcgga
     2401 taacaattcc cctgtagaaa taattttgtt taactttaat aaggagatat accatgggtg
     2461 gcggactaat agaagtggaa cgccctctgt acggagtaga ggtgtttccg ggtgaaacag
     2521 cccactttga aattgaactt tctgaacctg atgttcacgg ccagtggcgc ctgcgcggac
     2581 agcctttggc agcttcccct gactgtgaaa tcattgagga tggacgccgc catattctga
     2641 tccttcataa ctgtcagctg ggtatgacag gagaggtttc cttccaggct gctaataccc
     2701 gctctgcagc caatctgcgc gtgcgcgaat tgggtggagc aggaccgcca ccatatagtg
     2761 cagcaaacga cgaaaactac gctttagcag cgcatggtgg cgctcatacc tttaataacg
     2821 agaatgttag cgcacacaca ttcaacaatg aaaatgtcag taaacatacc tttaacaatg
     2881 aaaacgtgag ttgttattgc atcacgggag atgcactagt tgccctaccc gagggcgagt
     2941 cggtacgcat cgccgacatc gtgccgggtg cgcggcccaa cagtgacaac gccatcgacc
     3001 tgaaagtcct tgaccggcat ggcaatcccg tgctcgccga ccggctgttc cactccggcg
     3061 agcatccggt gtacacggtg cgtacggtcg aaggtctgcg tgtgacgggc accgcgaacc
     3121 acccgttgtt gtgtttggtc gacgtcgccg gggtgccgac cctgctgtgg aagctgatcg
     3181 acgaaatcaa gccgggcgat tacgcggtga ttcaacgcag cgcattcagc gtcgactgtg
     3241 caggttttgc ccgcgggaaa cccgaatttg cgcccacaac ctacacagtc ggcgtccctg
     3301 gactggtgcg tttcttggaa gcacaccacc gagacccgga cgcccaagct atcgccgacg
     3361 agctgaccga cgggcggttc tactacgcga aagtcgccag tgtcaccgac gccggcgtgc
     3421 agccggtgta tagccttcgt gtcgacacgg cagaccacgc gtttatcacg aacgggttcg
     3481 tcagccacgc tactggcctc accggtctga actcaggcct cacgacaaat cctggtgtat
     3541 ccgcttggca ggtcaacaca gcttatactg cgggacaatt ggtcacatat aacggcaaga
     3601 cgtataaatg tttgcagccc cacacctcct tggcaggatg ggaaccatcc aacgttcctg
     3661 ccttgtggca gcttcaataa ttaacctagg ctgctgccac cgctgagcaa taactagcat
     3721 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
     3781 ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     3841 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     3901 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     3961 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     4021 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     4081 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     4141 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     4201 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     4261 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     4321 atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa
     4381 tttattcata tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg
     4441 agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc
     4501 gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag
     4561 tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc
     4621 tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac
     4681 caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa
     4741 aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac
     4801 aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat
     4861 cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag
     4921 aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac
     4981 gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata
     5041 gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc
     5101 atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat
     5161 aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc
     5221 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     5281 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     5341 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     5401 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     5461 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     5521 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     5581 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     5641 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     5701 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     5761 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     5821 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     5881 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     5941 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     6001 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     6061 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag
     6121 tacaatctgc tctgat
//
