LOCUS       sequence-221559-        5916 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses Ulp1_R4 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: pCDB328
FEATURES             Location/Qualifiers
     source          1..5916
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     37..56
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     140..159
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        140..158
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    159..183
                     /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             198..220
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             233..250
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             920..937
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(990..1008)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1004..1051
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1088..1543
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1175..1194)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1385..1406
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1635..2450)
                     /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(1635..2450)
                     /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     2358..2377
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2442..2461
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2572..3160
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3061..3080
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3314..3331
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3346..3485
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3411..3430)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3571..3593
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3587..3778)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4587..5669)
                     /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     5631..5650
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5670..5747)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc
       61 agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga
      121 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat
      181 tcccctctag aaataatttt gtttaacttt aagaaggaga tataccatgg gccaccatca
      241 ccatcaccac ggttctctgg tgccggaact gaatgagaaa gacgatgacc aggttcagaa
      301 agcgctggag tcccgtgaaa acacccagct gatgaaccgt gacaacattg aaatcaccgt
      361 tcgtgacttc aaaactctgc gtccacgccg ttggctgaac gacaccatca tcgagttctt
      421 catgaagtac atcgagaaat ctactccgaa caccgttgcg ttcaactctt tcttctacac
      481 caacctgtct gagcgtggtt accagggcgt tcgtcgttgg atgaaacgta aaaagaccca
      541 gatcgacaag ctggacaaaa tcttcacccc aattaacctg aaccagtctc actgggctct
      601 cggtatcatt gacctgaaaa agaaaaccat tggctacgtg gactctctgt ctaatggtcc
      661 gaatgactcc tctaaacaga tcctgaccga cctccagaag tacgttaagg aagaatccaa
      721 gcacactatc ggcgaggact tcgacctgcg tcatctggac tgcccgcagc aaccaaacgg
      781 ttacgactgc ggtatctacg tttgcatgaa taccctgtac ggttctgcgg acgctccgct
      841 cgatttcgat caaaaagatg cggaacgtat gcgtcgcttc atcgcgcacc tcatcctcac
      901 cgacgcgctg aaactcgagc accaccacca ccaccactga gatccggctg ctaacaaagc
      961 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg
     1021 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc
     1081 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     1141 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     1201 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     1261 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     1321 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     1381 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     1441 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     1501 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
     1561 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     1621 tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata
     1681 tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca
     1741 ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca
     1801 acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca
     1861 ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact
     1921 tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta
     1981 ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta
     2041 caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca
     2101 cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg
     2161 agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat
     2221 tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg
     2281 ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca
     2341 cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg
     2401 gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt
     2461 gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga
     2521 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     2581 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     2641 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     2701 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     2761 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     2821 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     2881 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     2941 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     3001 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     3061 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     3121 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     3181 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     3241 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     3301 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     3361 tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc
     3421 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     3481 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     3541 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     3601 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     3661 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     3721 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     3781 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     3841 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     3901 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     3961 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     4021 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     4081 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     4141 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
     4201 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg
     4261 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     4321 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     4381 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     4441 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     4501 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     4561 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4621 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     4681 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     4741 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     4801 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     4861 accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     4921 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     4981 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     5041 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     5101 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     5161 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     5221 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     5281 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     5341 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     5401 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     5461 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     5521 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     5581 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     5641 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     5701 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     5761 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag
     5821 caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac
     5881 ggggcctgcc accataccca cgccgaaaca agcgct
//
