LOCUS       sequence-309526-        5610 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector for the de novo designed protein 
            Expression_vector_RO2_5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5610)
  AUTHORS   Pan X, Thompson MC, Zhang Y, Liu L, Fraser JS, Kelly MJS, Kortemme T
  TITLE     Expanding the space of protein geometries by computational design of
            de novo fold families.
  JOURNAL   Science. 2020 Aug 28;369(6507):1132-1136. doi: 
            10.1126/science.abc0881.
  PUBMED    32855341
REFERENCE   2  (bases 1 to 5610)
  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: "10.1126/science"; 
            journalName: "Science"; date: "2020-08-28- 28"; volume: "369"; 
            issue: "6507"; pages: "1132-1136"
COMMENT     Sequence Label: Expression_vector_RO2_5
FEATURES             Location/Qualifiers
     source          1..5610
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             475..492
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(545..563)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      559..606
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      643..1098
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(730..749)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     940..961
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1190..2005)
                     /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(1190..2005)
                     /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     1913..1932
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1997..2016
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2127..2715
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2616..2635
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2869..2886
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2901..3040
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2966..2985)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3126..3148
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3142..3333)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4142..5224)
                     /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     5186..5205
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5225..5302)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5508..5527
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg tgccgcgcgg cagccatatg ggtctcctcg taatcatcgt tagcaaggac
      181 aagaagttaa ttgaagaagc tcgcaagatg gccgagaagg caaatttatt gttaatcaca
      241 gcacctacgg acccacgcga gttggagact gcaatcaagt tacttcaaaa gtcaaatacg
      301 ccaatcaaga tcttaatctt atcagacgga acggacccca cggctgagaa gatcgcaaag
      361 aagttggcca aggaagcagc aacgaaggcc aatgccgagt accgcatccg caaggttact
      421 tcacccgacc aagcaaagcg ctggatcaag gagttctctg aggagtgact cgagcaccac
      481 caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
      541 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
      601 tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg
      661 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
      721 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
      781 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
      841 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
      901 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
      961 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     1021 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     1081 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     1141 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact
     1201 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     1261 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     1321 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     1381 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     1441 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     1501 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     1561 gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     1621 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
     1681 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     1741 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     1801 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     1861 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     1921 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag
     1981 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca
     2041 gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     2101 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     2161 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     2221 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     2281 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     2341 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     2401 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     2461 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     2521 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     2581 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     2641 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     2701 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     2761 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     2821 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     2881 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     2941 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     3001 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     3061 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     3121 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     3181 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     3241 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     3301 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     3361 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     3421 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     3481 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     3541 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     3601 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     3661 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     3721 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     3781 cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa
     3841 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac
     3901 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac
     3961 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc
     4021 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     4081 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     4141 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     4201 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     4261 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     4321 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     4381 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca
     4441 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     4501 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     4561 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     4621 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     4681 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     4741 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     4801 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     4861 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     4921 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     4981 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     5041 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     5101 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     5161 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     5221 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     5281 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     5341 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca
     5401 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg
     5461 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
     5521 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
     5581 tagaggatcg agatctcgat cccgcgaaat
//
