LOCUS       MZB116        5894 bp DNA     circular SYN 31-OCT-2023
DEFINITION  expresses Cth SUMO protease in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    pCDB302
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5894)
  AUTHORS   Lau Yue-Ting K., Vladimir Baytshtok, Tessa A. Howard, Brooke M. 
            Fiala, JayLee M. Morgan, Lauren P. Carter, David Baker, Christopher 
            D. Lima, Christopher D. Bahl
  TITLE     Discovery and engineering of enhanced SUMO protease enzymes
  JOURNAL   Journal of Biological Chemistry
REFERENCE   2  (bases 1 to 5894)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 8, 2023 from SnapGene 7.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Journal of 
            Biological Chemistry"
FEATURES             Location/Qualifiers
     source          1..5894
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     source          233..240
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     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)."
     primer_bind     185..232
                     /label=oMB284
     primer_bind     complement(185..227)
                     /label=oMB65
     RBS             200..222
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     211..261
                     /label=oMB462
     primer_bind     241..264
                     /label=oMB463
     unsure          246^247
                     /label=CPD Cleavage?
     misc_feature    247..909
                     /label=CPD
     CDS             880..909
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(889..925)
                     /label=oMB306
     primer_bind     908..938
                     /label=oMB307
     primer_bind     complement(966..984)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      980..1027
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1064..1519
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1151..1170)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1361..1382
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1611..2426)
                     /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     2334..2353
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2418..2437
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2548..3136
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3037..3056
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3290..3307
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3322..3461
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3387..3406)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3547..3569
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3563..3754)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4563..5645)
                     /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     5607..5626
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5646..5723)
                     /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 tgatttaaat
      241 agcctggcgg atggcaaaat tctgcataac cagaacgtga acagctgggg cccgattacc
      301 gtgaccccga ccaccgatgg cggcgaaacc cgctttgatg gccagattat tgtgcagatg
      361 gaaaacgatc cggtggtggc gaaagcggcg gcgaacctgg cgggcaaaca tgcggaaagc
      421 agcgtggtgg tgcagctgga tagcgatggc aactatcgcg tggtgtatgg cgatccgagc
      481 aaactggatg gcaaactgcg ctggcagctg gtgggccatg gccgcgatca tagcgaaacc
      541 aacaacaccc gcctgagcgg ctatagcgcg gatgaactgg cggtgaaact ggcgaaattt
      601 cagcagagct ttaaccaggc ggaaaacatt aacaacaaac cggatcatat tagcattgtg
      661 ggctgcagcc tggtgagcga tgataaacag aaaggctttg gccatcagtt tattaacgcg
      721 atggatgcga acggcctgcg cgtggatgtg agcgtgcgca gcagcgaact ggcggtggat
      781 gaagcgggcc gcaaacatac caaagatgcg aacggcgatt gggtgcagaa agcggaaaac
      841 aacaaagtga gcctgagctg ggatgcgcag ggcctggaac atcatcatca ccaccaccat
      901 catcatcatt aataaggatc cggctgctaa caaagcccga aaggaagctg agttggctgc
      961 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     1021 ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc
     1081 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     1141 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     1201 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     1261 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     1321 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     1381 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     1441 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     1501 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     1561 gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac
     1621 tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt
     1681 tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca
     1741 agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc
     1801 ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt
     1861 gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc
     1921 tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg
     1981 agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg
     2041 cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat
     2101 acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta
     2161 cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc
     2221 atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc
     2281 gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga
     2341 gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa
     2401 gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac
     2461 agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     2521 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     2581 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     2641 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     2701 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     2761 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     2821 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     2881 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     2941 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     3001 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     3061 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     3121 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     3181 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     3241 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     3301 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     3361 ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     3421 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     3481 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     3541 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     3601 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
     3661 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
     3721 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
     3781 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
     3841 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
     3901 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
     3961 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
     4021 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
     4081 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
     4141 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
     4201 gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga
     4261 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata
     4321 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga
     4381 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg
     4441 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     4501 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     4561 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     4621 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     4681 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     4741 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     4801 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatgtccgc accaacgcgc
     4861 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     4921 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     4981 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     5041 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
     5101 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     5161 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
     5221 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     5281 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     5341 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     5401 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     5461 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     5521 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     5581 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     5641 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     5701 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat
     5761 taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc
     5821 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc
     5881 gaaacaagcg ctca
//
