LOCUS       sequence-309527-        5631 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector for the de novo designed protein 
            Expression_vector_RO2_6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5631)
  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 5631)
  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_6
FEATURES             Location/Qualifiers
     source          1..5631
                     /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             496..513
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(566..584)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      580..627
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      664..1119
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(751..770)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     961..982
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1211..2026)
                     /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(1211..2026)
                     /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     1934..1953
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2018..2037
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2148..2736
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2637..2656
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2890..2907
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2922..3061
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2987..3006)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3147..3169
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3163..3354)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4163..5245)
                     /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     5207..5226
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5246..5323)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5529..5548
                     /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 gggctcgtag tcatcatcgt ttctaaggac
      181 aagaagttaa tcgaagaggc ccgcaagatg gccgagaagg ccaatttata cttattcacg
      241 ttagagccaa atgccgaccc aagtcaattg gacacgctcc gcaagtgggc ccaagagatc
      301 cttaagcgcg acgggccctc aaagctcaag gtattggttc tctcggacgg tacggacccc
      361 acagcacaaa agttggccaa gcttatcgca aagatcgtag caacggcagc caatgccgag
      421 gttcgcatcc gctccgtaac ttcacctgac caagccaagc gctggatcaa ggagttctcc
      481 gaggagtgac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
      541 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
      601 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
      661 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
      721 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
      781 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
      841 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
      901 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
      961 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     1021 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     1081 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     1141 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     1201 gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
     1261 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
     1321 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
     1381 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
     1441 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt
     1501 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
     1561 ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa
     1621 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
     1681 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     1741 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     1801 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     1861 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
     1921 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     1981 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat
     2041 tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt
     2101 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     2161 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     2221 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     2281 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     2341 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     2401 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     2461 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     2521 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     2581 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     2641 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     2701 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     2761 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     2821 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     2881 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     2941 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg
     3001 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     3061 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     3121 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     3181 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     3241 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     3301 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     3361 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     3421 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     3481 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
     3541 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
     3601 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
     3661 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
     3721 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
     3781 ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat
     3841 aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag
     3901 ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa
     3961 gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat
     4021 aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct
     4081 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     4141 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4201 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     4261 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     4321 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     4381 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     4441 agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     4501 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     4561 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     4621 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     4681 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     4741 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     4801 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     4861 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     4921 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     4981 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     5041 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     5101 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     5161 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     5221 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     5281 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
     5341 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
     5401 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
     5461 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
     5521 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg
     5581 ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa t
//
