LOCUS       sequence-450910-        6049 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TitinV15P-21-C-2-K-1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6049)
  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 6049)
  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: pAM100
FEATURES             Location/Qualifiers
     source          1..6049
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     8..25
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    40..179
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(105..124)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     265..287
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(281..472)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1281..2363)
                     /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     2325..2344
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2364..2441)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2487..2506
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2487..2505
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2506..2530
                     /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             2931..3524
                     /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             3555..3710
                     /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(3733..3751)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3747..3794
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3831..4286
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3918..3937)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4128..4149
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4378..5193)
                     /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(4378..5193)
                     /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     5101..5120
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5185..5204
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5315..5903
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5804..5823
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
       61 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
      121 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
      181 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
      241 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
      301 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
      361 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
      421 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
      481 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
      541 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
      601 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
      661 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
      721 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
      781 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
      841 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
      901 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa
      961 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     1021 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     1081 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     1141 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     1201 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     1261 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     1321 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     1381 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     1441 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     1501 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     1561 atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     1621 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     1681 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     1741 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     1801 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     1861 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     1921 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     1981 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     2041 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     2101 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     2161 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     2221 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     2281 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     2341 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     2401 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     2461 cttatgcgac tcctgcatta ggaaattaat acgactcact ataggggaat tgtgagcgga
     2521 taacaattcc cctgtagaaa taattttgtt taactttaat aaggagatat accatgggtg
     2581 gcggactaat agaagtggaa cgccctctgt acggagtaga ggtgtttccg ggtgaaacag
     2641 cccactttga aattgaactt tctgaacctg atgttcacgg ccagtggcgc ctgcgcggac
     2701 agcctttggc agcttcccct gactgtgaaa tcattgagga tggacgccgc catattctga
     2761 tccttcataa ctgtcagctg ggtatgacag gagaggtttc cttccaggct gctaataccc
     2821 gctctgcagc caatctgcgc gtgcgcgaat tgggtggagc aggaccgcca ccatatagtg
     2881 cagcaaacga cgaaaactac gctttagcag cgcattgtgg tggcaaatat tgcatcacgg
     2941 gagatgcact agttgcccta cccgagggcg agtcggtacg catcgccgac atcgtgccgg
     3001 gtgcgcggcc caacagtgac aacgccatcg acctgaaagt ccttgaccgg catggcaatc
     3061 ccgtgctcgc cgaccggctg ttccactccg gcgagcatcc ggtgtacacg gtgcgtacgg
     3121 tcgaaggtct gcgtgtgacg ggcaccgcga accacccgtt gttgtgtttg gtcgacgtcg
     3181 ccggggtgcc gaccctgctg tggaagctga tcgacgaaat caagccgggc gattacgcgg
     3241 tgattcaacg cagcgcattc agcgtcgact gtgcaggttt tgcccgcggg aaacccgaat
     3301 ttgcgcccac aacctacaca gtcggcgtcc ctggactggt gcgtttcttg gaagcacacc
     3361 accgagaccc ggacgcccaa gctatcgccg acgagctgac cgacgggcgg ttctactacg
     3421 cgaaagtcgc cagtgtcacc gacgccggcg tgcagccggt gtatagcctt cgtgtcgaca
     3481 cggcagacca cgcgtttatc acgaacgggt tcgtcagcca cgctactggc ctcaccggtc
     3541 tgaactcagg cctcacgaca aatcctggtg tatccgcttg gcaggtcaac acagcttata
     3601 ctgcgggaca attggtcaca tataacggca agacgtataa atgtttgcag ccccacacct
     3661 ccttggcagg atgggaacca tccaacgttc ctgccttgtg gcagcttcaa taattaacct
     3721 aggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
     3781 tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg
     3841 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     3901 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     3961 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     4021 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     4081 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     4141 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     4201 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     4261 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
     4321 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta
     4381 gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc
     4441 atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag
     4501 gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat
     4561 taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga
     4621 atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc
     4681 attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc
     4741 ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg
     4801 caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc
     4861 ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc
     4921 aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag
     4981 tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa
     5041 ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt
     5101 atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta atcgcggcct
     5161 agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta
     5221 agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     5281 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     5341 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     5401 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     5461 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     5521 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     5581 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     5641 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     5701 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     5761 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     5821 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     5881 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     5941 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     6001 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgac
//
