LOCUS       sequence-221554-        6003 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses Cth SUMO protease in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6003)
  AUTHORS   Lau YK, Baytshtok V, Howard TA, Fiala BM, Johnson JM, Carter LP, 
            Baker D, Lima CD, Bahl CD
  TITLE     Discovery and engineering of enhanced SUMO protease enzymes.
  JOURNAL   J Biol Chem. 2018 Aug 24;293(34):13224-13233. doi: 
            10.1074/jbc.RA118.004146. Epub 2018 Jul 5.
  PUBMED    29976752
REFERENCE   2  (bases 1 to 6003)
  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: 
            "10.1074/jbc.RA118.004146"; journalName: "J Biol Chem"; date: 
            "2018-08-24- 24"; volume: "293"; issue: "34"; pages: "13224-13233"
COMMENT     Sequence Label: pCDB302
FEATURES             Location/Qualifiers
     source          1..6003
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     35..54
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     142..161
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        142..160
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    165..181
                     /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)."
     RBS             200..222
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             989..1018
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(1075..1093)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1089..1136
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1173..1628
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1260..1279)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1470..1491
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1720..2535)
                     /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(1720..2535)
                     /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     2443..2462
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2527..2546
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2657..3245
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3146..3165
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3399..3416
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3431..3570
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3496..3515)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3656..3678
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3672..3863)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4672..5754)
                     /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     5716..5735
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5755..5832)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
       61 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
      121 tcgatctcga tcccgcgaaa ttaatacgac tcactatagg gaaattgtga gcggataaca
      181 attcccctct agaaataatt ttgtttaact ttaagaagga gatatacata tgtccggcct
      241 gcgtcctcct gcccgccagc tcatcacccc gctgtctgaa gaatggcgtt ctcgtgttga
      301 ggcggcacgt aacgccaacc cggctaccga actggcgaaa actctggaag gtcagccgct
      361 cgttcgccgc gacttcgagg aaaagctgct ccctgcgacc gcgtggctga acgacaacgt
      421 tatcatcggt gcgatcttct acatcgcgga ctacgttaac accaaaaaag gtgcgccgaa
      481 ccaggagccg aaatgcaccg cgttcacctc tttcttctgg cctcgtctgc tgtctcatgg
      541 tccgggtggt tgcggccgtc tcctccgccg tgccaacgtt cgtaaggcga acttcctgga
      601 tatcgacact atcctcatcc cgatttgcga atcttctcac tggaccctcg ccgttatccg
      661 tccaggtcgc cgtaccgttt ctcatctgga ctctatggct gcgggtcgtg gttctgagcg
      721 tgttaaagcg aaactgctgg aactggttaa attcgtactg gaagaccagt ttgttgaagc
      781 cgaatggcag gcggtggact tccaggcccc acgtcaaacc aacggttggg attgcggtgt
      841 ttttaccatc accaacgcga tctgtctggc gctgggcgtt gatcctgcgc aagcctacac
      901 cgaagcgcaa ctgccactgc agcgtcagcg tatcgctgcg gttctcctca atggcggttt
      961 caaaggtgat ttcacgctcg acgacctcca ccatcatcac caccatcacc atcaccacta
     1021 atgaggatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
     1081 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     1141 aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     1201 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     1261 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     1321 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     1381 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     1441 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     1501 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     1561 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     1621 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     1681 taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat
     1741 caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg
     1801 tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta
     1861 tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa
     1921 aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa
     1981 aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa
     2041 atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac
     2101 gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac
     2161 tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc
     2221 tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
     2281 cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt
     2341 aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt
     2401 cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata
     2461 cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg
     2521 ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt
     2581 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     2641 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     2701 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     2761 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     2821 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     2881 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     2941 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     3001 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     3061 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     3121 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     3181 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     3241 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     3301 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     3361 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     3421 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     3481 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     3541 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     3601 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     3661 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     3721 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     3781 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     3841 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     3901 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     3961 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     4021 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     4081 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     4141 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     4201 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     4261 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     4321 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     4381 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     4441 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     4501 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     4561 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     4621 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     4681 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4741 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     4801 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     4861 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     4921 gtcttcggta tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca gcccggactc
     4981 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     5041 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     5101 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     5161 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     5221 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     5281 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     5341 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     5401 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     5461 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     5521 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     5581 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     5641 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     5701 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     5761 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     5821 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     5881 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     5941 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     6001 tca
//
