LOCUS       sequence-386259-        6219 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  protein expression of the GST-tagged catalytic domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6219)
  TITLE     Knapp lab plasmids
REFERENCE   2  (bases 1 to 6219)
  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"
COMMENT     Sequence Label: pKM263-USP40
FEATURES             Location/Qualifiers
     source          1..6219
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1519..1538
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1622..1641
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1622..1640
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1672..1694
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1705..1722
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2380..2400
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(3561..3579)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3575..3622
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3797..3802,3803..3819,3820..3825))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3852..3870)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3938..4042
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4043..4111,4112..4903)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4261..4280)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5074..5662
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5563..5582
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5816..5833
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5848..5987
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5913..5932)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6073..6095
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(join(6089..6219,1..61))
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca
       61 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag
      121 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg
      181 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat
      241 gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga
      301 tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa
      361 cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt
      421 cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc
      481 agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggc caggacccaa
      541 cgctgcccga gatgcgccgc gtgcggctgc tggagatggc ggacgcgatg gatatgttct
      601 gccaagggtt ggtttgcgca ttcacagttc tccgcaagaa ttgattggct ccaattcttg
      661 gagtggtgaa tccgttagcg aggtgccgcc ggcttccatt caggtcgagg tggcccggct
      721 ccatgcaccg cgacgcaacg cggggaggca gacaaggtat agggcggcgc ctacaatcca
      781 tgccaacccg ttccatgtgc tcgccgaggc ggcataaatc gccgtgacga tcagcggtcc
      841 aatgatcgaa gttaggctgg taagagccgc gagcgatcct tgaagctgtc cctgatggtc
      901 gtcatctacc tgcctggaca gcatggcctg caacgcgggc atcccgatgc cgccggaagc
      961 gagaagaatc ataatgggga aggccatcca gcctcgcgtc gcgaacgcca gcaagacgta
     1021 gcccagcgcg tcggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
     1081 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
     1141 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
     1201 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     1261 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     1321 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
     1381 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
     1441 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
     1501 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
     1561 gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa
     1621 ttaatacgac tcactatagg gagaccacaa cggtttccct ctagaaataa ttttgtttaa
     1681 ctttaagaag gagatataca tatgcaccat caccatcacc atgctagctt catgtcccct
     1741 atactaggtt attggaaaat taagggcctt gtgcaaccca ctcgacttct tttggaatat
     1801 cttgaagaaa aatatgaaga gcatttgtat gagcgcgatg aaggtgataa atggcgaaac
     1861 aaaaagtttg aattgggttt ggagtttccc aatcttcctt attatattga tggtgatgtt
     1921 aaattaacac agtctatggc catcatacgt tatatagctg acaagcacaa catgttgggt
     1981 ggttgtccaa aagagcgtgc agagatttca atgcttgaag gagcggtttt ggatattaga
     2041 tacggtgttt cgagaattgc atatagtaaa gactttgaaa ctctcaaagt tgattttctt
     2101 agcaagctac ctgaaatgct gaaaatgttc gaagatcgtt tatgtcataa aacatattta
     2161 aatggtgatc atgtaaccca tcctgacttc atgttgtatg acgctcttga tgttgtttta
     2221 tacatggacc caatgtgcct ggatgcgttc ccaaaattag tttgttttaa aaaacgtatt
     2281 gaagctatcc cacaaattga taagtacttg aaatccagca agtatatagc atggcctttg
     2341 cagggctggc aagccacgtt tggtggtggc gaccatctcg agaatcttta ttttcagggc
     2401 gccatgggcc gtgagtttac caacctgagc ggtatccgta accagggtgg cacctgctac
     2461 ctgaacagcc tgctgcaaac cctgcacttt accccggagt tccgtgaagc gctgtttagc
     2521 ctgggtccgg aggaactggg cctgttcgag gacaaggata aaccggacgc gaaagtgcgt
     2581 atcattccgc tgcagctgca acgtctgttt gcgcagctgc tgctgctgga tcaagaagcg
     2641 gcgagcaccg cggacctgac cgatagcttc ggttggacca gcaacgagga aatgcgtcag
     2701 cacgacgtgc aagagctgaa ccgtatcctg tttagcgcgc tggaaaccag cctggttggc
     2761 accagcggcc acgatctgat ttaccgtctg tatcacggca ccatcgtgaa ccagattgtt
     2821 tgcaaggagt gcaaaaacgt gagcgagcgt caagaagact ttctggatct gaccgtggcg
     2881 gttaagaacg ttagcggtct ggaagacgcg ctgtggaaca tgtacgtgga ggaagaggtt
     2941 ttcgactgcg ataacctgta tcactgcggc acctgcgatc gtctggtgaa ggcggcgaaa
     3001 agcgcgaagc tgcgtaaact gccgccgttc ctgaccgtga gcctgctgcg tttcaacttt
     3061 gacttcgtta agtgcgagcg ttacaaagaa accagctgct atacctttcc gctgcgtatc
     3121 aacctgaaac cgttctgcga gcagagcgaa ctggacgatc tggaatacat ctacgatctg
     3181 tttagcgtta tcattcacaa gggtggctgc tacggtggcc actaccacgt gtatatcaaa
     3241 gacgttgatc acctgggtaa ctggcagttc caagaagaga agagcaaacc ggacgtgaac
     3301 ctgaaggatc tgcagagcga agaggaaatc gaccacccgc tgatgatcct gaaagcgatt
     3361 ctgctggagg aaaacaacct gattccggtt gatcagctgg gtcaaaaact gctgaagaaa
     3421 atcggcatta gctggaacaa gaaatatcgt aagcagtaag gatccggctg ctaacgcatt
     3481 agctggaaca agaaatatcg taagcagtaa ggatccggct gctaacaaag cccgaaagga
     3541 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     3601 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggatatc ccgcaagagg
     3661 cccggcagta ccggcataac caagcctatg cctacagcat ccagggtgac ggtgccgagg
     3721 atgacgatga gcgcattgtt agatttcata cacggtgcct gactgcgtta gcaatttaac
     3781 tgtgataaac taccgcatta aagcttatcg atgataagct gtcaaacatg agaattcttg
     3841 aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     3901 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     3961 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     4021 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     4081 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     4141 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     4201 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     4261 gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat
     4321 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     4381 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     4441 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     4501 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     4561 cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac
     4621 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     4681 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     4741 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     4801 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     4861 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     4921 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     4981 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     5041 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     5101 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     5161 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     5221 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     5281 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     5341 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     5401 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     5461 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     5521 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     5581 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     5641 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     5701 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     5761 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     5821 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
     5881 cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     5941 agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc
     6001 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     6061 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     6121 cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct
     6181 gttcatccgc gtccagctcg ttgagtttct ccagaagcg
//
