LOCUS       sequence-198445-        5545 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5545)
  AUTHORS   Sims JJ, Scavone F, Cooper EM, Kane LA, Youle RJ, Boeke JD, Cohen RE
  TITLE     Polyubiquitin-sensor proteins reveal localization and linkage-type 
            dependence of cellular ubiquitin signaling.
  JOURNAL   Nat Methods. 2012 Feb 5. doi: 10.1038/nmeth.1888.
  PUBMED    22306808
REFERENCE   2  (bases 1 to 5545)
  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"; journalName: "Nat 
            Methods. 2012 Feb 5. doi: 10.1038/nmeth.1888."
COMMENT     Sequence Label: pET28a-Rx3(A7)
FEATURES             Location/Qualifiers
     source          1..5545
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    13..37
                     /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             52..74
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             93..110
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             120..137
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             403..420
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(473..491)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      487..534
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      571..1026
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(658..677)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     868..889
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1118..1933)
                     /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(1118..1933)
                     /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     1841..1860
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1925..1944
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2055..2643
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2544..2563
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2797..2814
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2829..2968
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2894..2913)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3054..3076
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3070..3261)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4070..5152)
                     /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     5114..5133
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5153..5230)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5436..5455
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(5539..5545,1..13)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5539..5545,1..12)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 actcactata ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa
       61 ctttaagaag gagatatacc atgcccagca gccatcatca tcatcatcac agcagcggcc
      121 tggtgccgcg cggcagccat atgaccgagg aggagcaatt cgcactcgca ctgaagatga
      181 gcgagcaaga ggcggcggct gccgctgctg ccgcagaaga ggaagaagaa cagtttgctc
      241 tggctctcaa aatgagtgaa caggaagctg ctgccgcagc ggcggctgcc gaggaggaag
      301 aagagctctt gaggaaagcc attgcagaaa gtctgaatag ttgccggcct tcttgaggat
      361 ccgaattcga gctccgtcga caagcttgcg gccgcactcg agcaccacca ccaccaccac
      421 tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
      481 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
      541 ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
      601 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
      661 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
      721 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
      781 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
      841 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
      901 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
      961 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     1021 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     1081 aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca
     1141 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
     1201 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
     1261 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
     1321 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa
     1381 gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
     1441 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc
     1501 gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg
     1561 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg
     1621 ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct
     1681 tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa
     1741 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc
     1801 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc
     1861 catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt
     1921 gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc
     1981 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     2041 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     2101 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     2161 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     2221 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     2281 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     2341 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     2401 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     2461 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     2521 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     2581 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     2641 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     2701 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     2761 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     2821 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat
     2881 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     2941 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     3001 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     3061 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     3121 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     3181 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
     3241 ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt
     3301 aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc
     3361 ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag
     3421 aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg
     3481 tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg
     3541 cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc
     3601 agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta
     3661 accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg
     3721 cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc
     3781 gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag
     3841 gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc
     3901 cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt
     3961 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     4021 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     4081 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     4141 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     4201 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     4261 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     4321 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
     4381 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     4441 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     4501 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     4561 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     4621 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     4681 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     4741 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     4801 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     4861 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     4921 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     4981 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     5041 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     5101 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     5161 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     5221 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc
     5281 agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg
     5341 gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc
     5401 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca
     5461 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag
     5521 atctcgatcc cgcgaaatta atacg
//
