LOCUS       sequence-221696-        5916 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses Ulp1_R3 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: pCDB327
FEATURES             Location/Qualifiers
     source          1..5916
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     75..94
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     178..197
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        178..196
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    197..221
                     /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             236..258
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             271..288
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             958..975
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1028..1046)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1042..1089
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1126..1581
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1213..1232)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1423..1444
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1673..2488)
                     /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(1673..2488)
                     /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     2396..2415
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2480..2499
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2610..3198
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3099..3118
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3352..3369
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3384..3523
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3449..3468)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3609..3631
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3625..3816)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4625..5707)
                     /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     5669..5688
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5708..5785)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
       61 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
      121 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa
      181 tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt
      241 ttaactttaa gaaggagata taccatgggc caccatcacc atcaccacgg ttctctggtg
      301 ccagagctga acgaaaagga cgacgaccag gttcagaaag cgctcgattc tcgcgaaaac
      361 acccagctga tgaaccgtga caacatcgaa atcaccgtgc gtgacttcaa aaccctggag
      421 ccgcgtcgct ggctcaacga caccatcatc gagtttttca tgaagtacat cgagaagtcc
      481 accccgaaca ctgtggcgtt caactctttc ttctacacca acctctccga acgtggttac
      541 cagggtgtgc gtcgttggat gaagcgcaaa aaaacccaga tcgacaaact ggacaaaatc
      601 ttcactccga tcaacctgaa ccagtctcac tgggcgctgg gcattattga cctgaaaaag
      661 aaaaccattg gctacgtgga ctctctctcc aacggtccaa acgactcctc taagcagatc
      721 ctgaccgacc tgcagaaata cgttgaagaa gaatctaaac acaccatcgg cgaagacttt
      781 gacctccgtc acctcgactg cccgcagcag ccaaacggtt atgattgcgg tatctatgtt
      841 tgcatgaaca ccctctacgg ttctgcggac gctccgctcg acttcgactc taaagacgct
      901 gagcgtatgc gccgtttcat cgcgcacctg atcctcaccg acgcgctgaa actcgagcac
      961 caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct
     1021 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     1081 ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg
     1141 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     1201 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     1261 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     1321 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     1381 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     1441 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     1501 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     1561 aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat
     1621 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa
     1681 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
     1741 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
     1801 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
     1861 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
     1921 gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac
     1981 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
     2041 cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
     2101 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta
     2161 atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag
     2221 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
     2281 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
     2341 gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc
     2401 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc
     2461 aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag
     2521 acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     2581 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     2641 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     2701 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     2761 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     2821 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     2881 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     2941 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     3001 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     3061 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     3121 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     3181 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     3241 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     3301 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     3361 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     3421 atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     3481 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     3541 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     3601 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     3661 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     3721 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     3781 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     3841 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     3901 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     3961 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     4021 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     4081 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     4141 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     4201 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     4261 gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc
     4321 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     4381 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     4441 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     4501 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     4561 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     4621 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     4681 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     4741 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     4801 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     4861 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc
     4921 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     4981 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     5041 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     5101 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     5161 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     5221 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     5281 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     5341 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     5401 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     5461 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     5521 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     5581 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     5641 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     5701 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     5761 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     5821 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     5881 gcatgcaagg agatggcgcc caacagtccc ccggcc
//
