LOCUS       sequence-212915-        6605 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Bacterial Expression of RNF144A RBR domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6605)
  AUTHORS   Michel MA, Swatek KN, Hospenthal MK, Komander D
  TITLE     Ubiquitin Linkage-Specific Affimers Reveal Insights into K6-Linked 
            Ubiquitin Signaling.
  JOURNAL   Mol Cell. 2017 Oct 5;68(1):233-246.e5. doi: 
            10.1016/j.molcel.2017.08.020. Epub 2017 Sep 21.
  PUBMED    28943312
REFERENCE   2  (bases 1 to 6605)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.molcel.2017.08.020"; journalName: "Mol Cell"; date: 
            "2017-10-5- 5"; volume: "68"; issue: "1"; pages: "233-246.e5"
COMMENT     Sequence Label: Human RNF144A (16-228)
FEATURES             Location/Qualifiers
     source          1..6605
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     106..125
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     209..228
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        209..227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    228..252
                     /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             267..289
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             302..319
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             320..973
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             992..1015
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1678..1695
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1748..1766)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1762..1809
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1846..2301
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1933..1952)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2143..2164
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2393..3208)
                     /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(2393..3208)
                     /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     3116..3135
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3200..3219
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3330..3918
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3819..3838
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4072..4089
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4104..4243
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4169..4188)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4329..4351
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4345..4536)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5345..6427)
                     /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     6389..6408
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6428..6505)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat ataccatggc
      301 acatcaccat caccatcaca tgtcccctat actaggttat tggaaaatta agggccttgt
      361 gcaacccact cgacttcttt tggaatatct tgaagaaaaa tatgaagagc atttgtatga
      421 gcgcgatgaa ggtgataaat ggcgaaacaa aaagtttgaa ttgggtttgg agtttcccaa
      481 tcttccttat tatattgatg gtgatgttaa attaacacag tctatggcca tcatacgtta
      541 tatagctgac aagcacaaca tgttgggtgg ttgtccaaaa gagcgtgcag agatttcaat
      601 gcttgaagga gcggttttgg atattagata cggtgtttcg agaattgcat atagtaaaga
      661 ctttgaaact ctcaaagttg attttcttag caagctacct gaaatgctga aaatgttcga
      721 agatcgttta tgtcataaaa catatttaaa tggtgatcat gtaacccatc ctgacttcat
      781 gttgtatgac gctcttgatg ttgttttata catggaccca atgtgcctgg atgcgttccc
      841 aaaattagtt tgttttaaaa aacgtattga agctatccca caaattgata agtacttgaa
      901 atccagcaag tatatagcat ggcctttgca gggctggcaa gccacgtttg gtggtggcga
      961 ccatcctcca aaatcggatc tgagcagcgg tctggaagtt ctgtttcagg gcccgccgct
     1021 ggtgtcttgc aagctctgtc ttggggagta cccagtggag cagatgacaa ccatagccca
     1081 gtgccaatgc atcttctgta ctctgtgcct gaaacagtat gttgagctct tgatcaaaga
     1141 aggattagaa accgcaatta gctgcccaga tgctgcctgc cctaaacagg gccacctaca
     1201 ggagaacgag attgagtgca tggttgcagc tgaaattatg caaagatata aaaagctaca
     1261 atttgaaaga gaggtgctgt ttgatccctg tcggacttgg tgcccggcgt ccacctgcca
     1321 agctgtgtgt cagctccagg acgtggggct gcagaccccc cagccagtgc agtgcaaagc
     1381 ctgccgtatg gaattctgct ccacctgcaa agccagctgg caccctggcc agggctgccc
     1441 ggagaccatg ccgatcacct tcctccccgg ggagaccagt gctgctttca aaatggaaga
     1501 agatgacgcg cccatcaagc gctgccccaa gtgcaaagtc tacatcgagc gagacgaagg
     1561 ctgcgcgcag atgatgtgca agaactgcaa gcacgccttc tgctggtact gcctggagtc
     1621 tctggacgat gatttccttc tgatacacta cgattaaagc tttctagacc atttaaacac
     1681 caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct
     1741 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     1801 ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg
     1861 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     1921 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     1981 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     2041 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     2101 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     2161 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     2221 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     2281 aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat
     2341 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa
     2401 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
     2461 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
     2521 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
     2581 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
     2641 gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac
     2701 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
     2761 cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
     2821 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta
     2881 atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag
     2941 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
     3001 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
     3061 gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc
     3121 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc
     3181 aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag
     3241 acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     3301 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     3361 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     3421 acaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     3481 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     3541 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     3601 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     3661 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     3721 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     3781 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     3841 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     3901 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     3961 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     4021 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     4081 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     4141 atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     4201 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     4261 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     4321 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     4381 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     4441 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     4501 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     4561 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     4621 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     4681 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     4741 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     4801 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     4861 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     4921 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     4981 gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc
     5041 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     5101 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     5161 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     5221 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     5281 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     5341 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     5401 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     5461 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     5521 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     5581 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc
     5641 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     5701 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     5761 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     5821 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     5881 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     5941 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     6001 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     6061 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     6121 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     6181 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     6241 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     6301 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     6361 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     6421 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     6481 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     6541 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     6601 gcatg
//
