LOCUS       sequence-450917-        6076 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TitinV15P-23-K-9-C-1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6076)
  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 6076)
  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: pAM99
FEATURES             Location/Qualifiers
     source          1..6076
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     400..419
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     484..503
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      614..1202
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1103..1122
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1356..1373
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1388..1527
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1453..1472)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1613..1635
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1629..1820)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2629..3711)
                     /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     3673..3692
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3712..3789)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3835..3854
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3835..3853
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3854..3878
                     /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             4306..4899
                     /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             4930..5085
                     /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(5108..5126)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5122..5169
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5206..5661
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5293..5312)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5503..5524
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(5753..6076,1..492))
                     /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(join(5753..6076,1..492))
                     /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"
ORIGIN
        1 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg
       61 atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc
      121 cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt
      181 tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt
      241 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac
      301 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc
      361 atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc
      421 atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg
      481 aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca
      541 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
      601 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
      661 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
      721 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
      781 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
      841 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
      901 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
      961 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     1021 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     1081 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     1141 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     1201 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     1261 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     1321 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     1381 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg
     1441 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     1501 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     1561 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     1621 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc
     1681 tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg
     1741 ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg
     1801 gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta
     1861 atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc
     1921 cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga
     1981 aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt
     2041 agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc
     2101 gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca
     2161 gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa
     2221 ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc
     2281 acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg
     2341 ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg
     2401 ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc
     2461 ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc
     2521 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     2581 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     2641 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     2701 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     2761 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     2821 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     2881 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
     2941 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     3001 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     3061 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     3121 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     3181 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     3241 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     3301 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     3361 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     3421 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     3481 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     3541 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     3601 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     3661 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     3721 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     3781 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aaattaatac
     3841 gactcactat aggggaattg tgagcggata acaattcccc tgtagaaata attttgttta
     3901 actttaataa ggagatatac catgggtggc ggactaatag aagtggaacg ccctctgtac
     3961 ggagtagagg tgtttccggg tgaaacagcc cactttgaaa ttgaactttc tgaacctgat
     4021 gttcacggcc agtggcgcct gcgcggacag cctttggcag cttcccctga ctgtgaaatc
     4081 attgaggatg gacgccgcca tattctgatc cttcataact gtcagctggg tatgacagga
     4141 gaggtttcct tccaggctgc taatacccgc tctgcagcca atctgcgcgt gcgcgaattg
     4201 ggtggagcag gaccgccacc atatagtgca gcaaacgacg aaaactacgc tttagcagcg
     4261 catggtggca aacacacatt caacaatgaa aatgtcagtt gttattgcat cacgggagat
     4321 gcactagttg ccctacccga gggcgagtcg gtacgcatcg ccgacatcgt gccgggtgcg
     4381 cggcccaaca gtgacaacgc catcgacctg aaagtccttg accggcatgg caatcccgtg
     4441 ctcgccgacc ggctgttcca ctccggcgag catccggtgt acacggtgcg tacggtcgaa
     4501 ggtctgcgtg tgacgggcac cgcgaaccac ccgttgttgt gtttggtcga cgtcgccggg
     4561 gtgccgaccc tgctgtggaa gctgatcgac gaaatcaagc cgggcgatta cgcggtgatt
     4621 caacgcagcg cattcagcgt cgactgtgca ggttttgccc gcgggaaacc cgaatttgcg
     4681 cccacaacct acacagtcgg cgtccctgga ctggtgcgtt tcttggaagc acaccaccga
     4741 gacccggacg cccaagctat cgccgacgag ctgaccgacg ggcggttcta ctacgcgaaa
     4801 gtcgccagtg tcaccgacgc cggcgtgcag ccggtgtata gccttcgtgt cgacacggca
     4861 gaccacgcgt ttatcacgaa cgggttcgtc agccacgcta ctggcctcac cggtctgaac
     4921 tcaggcctca cgacaaatcc tggtgtatcc gcttggcagg tcaacacagc ttatactgcg
     4981 ggacaattgg tcacatataa cggcaagacg tataaatgtt tgcagcccca cacctccttg
     5041 gcaggatggg aaccatccaa cgttcctgcc ttgtggcagc ttcaataatt aacctaggct
     5101 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     5161 ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg
     5221 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     5281 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     5341 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     5401 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     5461 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     5521 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     5581 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     5641 aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat
     5701 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa
     5761 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
     5821 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
     5881 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
     5941 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
     6001 gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac
     6061 gctcgtcatc aaaatc
//
