LOCUS       sequence-450909-        6118 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TitinV15P-23-K-9-C-15.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6118)
  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 6118)
  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: pAM98
FEATURES             Location/Qualifiers
     source          1..6118
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(13..90)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     136..155
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        136..154
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    155..179
                     /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             649..1242
                     /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             1273..1428
                     /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(1451..1469)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1465..1512
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1549..2004
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1636..1655)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1846..1867
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2096..2911)
                     /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(2096..2911)
                     /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     2819..2838
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2903..2922
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3033..3621
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3522..3541
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3775..3792
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3807..3946
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3872..3891)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4032..4054
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4048..4239)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5048..6118,1..12))
                     /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     6092..6111
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
       61 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact
      121 cctgcattag gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc
      181 ctgtagaaat aattttgttt aactttaata aggagatata ccatgggtgg cggactaata
      241 gaagtggaac gccctctgta cggagtagag gtgtttccgg gtgaaacagc ccactttgaa
      301 attgaacttt ctgaacctga tgttcacggc cagtggcgcc tgcgcggaca gcctttggca
      361 gcttcccctg actgtgaaat cattgaggat ggacgccgcc atattctgat ccttcataac
      421 tgtcagctgg gtatgacagg agaggtttcc ttccaggctg ctaatacccg ctctgcagcc
      481 aatctgcgcg tgcgcgaatt gggtggagca ggaccgccac catatagtgc agcaaacgac
      541 gaaaactacg ctttagcagc gcatggtggc aaacacacat tcaacaatga aaatgtcagt
      601 tgtaggctcg ggggagcggc tagcattgct gtgcaagcgc cagcatattg catcacggga
      661 gatgcactag ttgccctacc cgagggcgag tcggtacgca tcgccgacat cgtgccgggt
      721 gcgcggccca acagtgacaa cgccatcgac ctgaaagtcc ttgaccggca tggcaatccc
      781 gtgctcgccg accggctgtt ccactccggc gagcatccgg tgtacacggt gcgtacggtc
      841 gaaggtctgc gtgtgacggg caccgcgaac cacccgttgt tgtgtttggt cgacgtcgcc
      901 ggggtgccga ccctgctgtg gaagctgatc gacgaaatca agccgggcga ttacgcggtg
      961 attcaacgca gcgcattcag cgtcgactgt gcaggttttg cccgcgggaa acccgaattt
     1021 gcgcccacaa cctacacagt cggcgtccct ggactggtgc gtttcttgga agcacaccac
     1081 cgagacccgg acgcccaagc tatcgccgac gagctgaccg acgggcggtt ctactacgcg
     1141 aaagtcgcca gtgtcaccga cgccggcgtg cagccggtgt atagccttcg tgtcgacacg
     1201 gcagaccacg cgtttatcac gaacgggttc gtcagccacg ctactggcct caccggtctg
     1261 aactcaggcc tcacgacaaa tcctggtgta tccgcttggc aggtcaacac agcttatact
     1321 gcgggacaat tggtcacata taacggcaag acgtataaat gtttgcagcc ccacacctcc
     1381 ttggcaggat gggaaccatc caacgttcct gccttgtggc agcttcaata attaacctag
     1441 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     1501 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
     1561 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     1621 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     1681 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     1741 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     1801 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     1861 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     1921 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     1981 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     2041 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga
     2101 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat
     2161 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga
     2221 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta
     2281 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat
     2341 ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat
     2401 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct
     2461 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca
     2521 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt
     2581 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag
     2641 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc
     2701 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact
     2761 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat
     2821 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag
     2881 agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag
     2941 cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     3001 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     3061 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     3121 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     3181 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     3241 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     3301 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     3361 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     3421 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     3481 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     3541 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     3601 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     3661 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     3721 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     3781 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     3841 ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     3901 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     3961 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     4021 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     4081 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg
     4141 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa
     4201 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg
     4261 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt
     4321 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg
     4381 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga
     4441 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt
     4501 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca
     4561 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat
     4621 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga
     4681 caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc
     4741 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc
     4801 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa
     4861 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat
     4921 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc
     4981 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
     5041 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     5101 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     5161 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     5221 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     5281 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     5341 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     5401 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     5461 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     5521 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     5581 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     5641 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     5701 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     5761 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     5821 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     5881 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     5941 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     6001 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     6061 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgt
//
