LOCUS       sequence-221565-        5916 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses Ulp1_R1 in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5916)
  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 5916)
  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: pCDB325
FEATURES             Location/Qualifiers
     source          1..5916
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     74..93
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     177..196
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        177..195
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    196..220
                     /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             235..257
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             270..287
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             957..974
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1027..1045)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1041..1088
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1125..1580
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1212..1231)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1422..1443
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1672..2487)
                     /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(1672..2487)
                     /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     2395..2414
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2479..2498
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2609..3197
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3098..3117
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3351..3368
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3383..3522
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3448..3467)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3608..3630
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3624..3815)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4624..5706)
                     /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     5668..5687
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5707..5784)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
       61 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
      121 tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat
      181 acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt
      241 taactttaag aaggagatat accatgggcc accatcacca tcaccacggt tctctggttc
      301 cagaactgaa cgagaaagac gacgaccagg ttcagaaagc gctggactcc cgtgaaaaca
      361 cccagctgat gaaccgtgac aatatcgaaa tcaccgttcg tgacttcaaa accctcgaac
      421 cgcgtcgctg gctcaacgac accatcatcg aattcttcat gaagtacatc gagaagtcca
      481 ccccgaacac tgtggcgttc aactccttct tttacactaa cctgtctgaa cgtggttacc
      541 agggcgttcg ccgttggatg aaacgcaaaa aaacccagat cgacaagctc gacaaaatct
      601 tcactccgat caacctgaat cagtctcact gggcgctggg tatcattgac ctgaagaaaa
      661 agaccattgg ctacgttgat tctctgtcta acggtccgaa cgactcttcc aaacagatcc
      721 tgaccgatct gcaaaagtac gtgaaggagg aatccaagca cactatcggc gaagacttcg
      781 acctgcgtca cctcgactgc ccgcagcagc cgaacggcta cgattgcggt atctacgtgt
      841 gcatgaatac cctgtacggt tctgctgatg cgccactcga ttttgaccaa aaagatgcgg
      901 aacgtatgcg tcgttttatc gctcacctga ttctgactga tgctctgaaa ctcgagcacc
      961 accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     1021 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     1081 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     1141 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     1201 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     1261 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     1321 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     1381 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     1441 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     1501 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     1561 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     1621 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa
     1681 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt
     1741 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc
     1801 aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt
     1861 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg
     1921 tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg
     1981 ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc
     2041 gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg
     2101 gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa
     2161 tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt
     2221 acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac
     2281 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg
     2341 cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg
     2401 agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca
     2461 agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga
     2521 cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     2581 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     2641 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     2701 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     2761 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     2821 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     2881 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     2941 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     3001 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     3061 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     3121 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     3181 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     3241 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     3301 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     3361 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     3421 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     3481 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     3541 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     3601 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     3661 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     3721 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     3781 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
     3841 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     3901 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
     3961 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     4021 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     4081 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     4141 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     4201 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     4261 agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg
     4321 aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat
     4381 accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg
     4441 acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt
     4501 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
     4561 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg
     4621 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4681 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
     4741 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
     4801 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
     4861 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg
     4921 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
     4981 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
     5041 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
     5101 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
     5161 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
     5221 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
     5281 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
     5341 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
     5401 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
     5461 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
     5521 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
     5581 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
     5641 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
     5701 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
     5761 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
     5821 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
     5881 catgcaagga gatggcgccc aacagtcccc cggcca
//
