LOCUS       sequence-309535-        5595 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector for the de novo designed protein 
            Expression_vector_RO2_20.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5595)
  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 5595)
  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_20
FEATURES             Location/Qualifiers
     source          1..5595
                     /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             460..477
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(530..548)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      544..591
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      628..1083
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(715..734)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     925..946
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1175..1990)
                     /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(1175..1990)
                     /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     1898..1917
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1982..2001
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2112..2700
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2601..2620
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2854..2871
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2886..3025
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2951..2970)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3111..3133
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3127..3318)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4127..5209)
                     /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     5171..5190
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5210..5287)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5493..5512
                     /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 ggattattgg tcctcatttg gtcgaatgac
      181 aagaagctca tcgaggaagc tcgcaagatg gctgagaagg ccaatctcta cctcttgacg
      241 ctcgaaactg acgacaagaa gatcgaggac atcttaaagt cgctcgggcc gcccgttaag
      301 atcctcgttc tcttagagga cacaaaggac gccgacaagg tcaagaagga gatcgagaag
      361 aaggcccgca agaagaattt acccgtacgc atccgcaagg taacttcgcc agacgaggca
      421 aagcgctgga tcaaggagtt cagtgaggag tgactcgagc accaccacca ccaccactga
      481 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
      541 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
      601 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
      661 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
      721 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
      781 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
      841 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
      901 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
      961 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     1021 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     1081 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     1141 acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat
     1201 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
     1261 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
     1321 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
     1381 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt
     1441 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
     1501 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat
     1561 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
     1621 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
     1681 tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
     1741 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
     1801 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     1861 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     1921 ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa
     1981 tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg
     2041 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     2101 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     2161 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     2221 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     2281 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     2341 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     2401 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     2461 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     2521 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     2581 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     2641 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     2701 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     2761 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     2821 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     2881 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
     2941 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     3001 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     3061 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     3121 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     3181 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     3241 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
     3301 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     3361 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     3421 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     3481 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     3541 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     3601 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     3661 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     3721 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     3781 ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     3841 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     3901 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     3961 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     4021 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     4081 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     4141 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4201 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     4261 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     4321 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     4381 cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa
     4441 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     4501 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     4561 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     4621 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     4681 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     4741 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     4801 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     4861 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     4921 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     4981 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     5041 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     5101 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     5161 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     5221 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     5281 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
     5341 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
     5401 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
     5461 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     5521 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc
     5581 tcgatcccgc gaaat
//
