LOCUS       sequence-309540-        5604 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector for the de novo designed protein 
            Expression_vector_RO2_25.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5604)
  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 5604)
  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_25
FEATURES             Location/Qualifiers
     source          1..5604
                     /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             469..486
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(539..557)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      553..600
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      637..1092
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(724..743)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     934..955
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1184..1999)
                     /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(1184..1999)
                     /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     1907..1926
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1991..2010
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2121..2709
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2610..2629
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2863..2880
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2895..3034
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2960..2979)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3120..3142
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3136..3327)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4136..5218)
                     /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     5180..5199
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5219..5296)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5502..5521
                     /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 acgcttttcg tactcatctt atcaaatgac
      181 aagaagctta tcgaggaagc ccgcaagatg gcagagaagg ccaatttgat cctcatcacg
      241 gtcggggacg aagaggagtt aaagaaggcc atcaagaagg ccgacgacat cgctaagaag
      301 caaaattcgt cagaggccaa gatcctcatc ttgcttgaga agccagtctc gcctgagtac
      361 gagaagaagt tacaaaagta cgcagacgca gaggttcgcg ttcgcacagt tacgtcacca
      421 gacgaggcca agcgctggat caaggagttc tctgaggagt gactcgagca ccaccaccac
      481 caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
      541 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
      601 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
      661 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
      721 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
      781 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
      841 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
      901 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
      961 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     1021 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     1081 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     1141 tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga
     1201 gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa
     1261 gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct
     1321 ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt
     1381 caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg
     1441 gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat
     1501 caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa
     1561 atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga
     1621 acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga
     1681 atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa
     1741 aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat
     1801 ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg
     1861 gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt
     1921 tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt
     1981 cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta
     2041 ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     2101 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     2161 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     2221 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     2281 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     2341 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     2401 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     2461 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     2521 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     2581 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     2641 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     2701 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     2761 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     2821 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     2881 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     2941 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     3001 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     3061 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     3121 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     3181 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     3241 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     3301 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     3361 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     3421 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     3481 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     3541 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     3601 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     3661 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     3721 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     3781 catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt
     3841 ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag
     3901 cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag
     3961 cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac
     4021 gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     4081 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
     4141 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     4201 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg
     4261 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
     4321 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
     4381 agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg
     4441 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
     4501 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
     4561 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
     4621 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
     4681 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
     4741 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
     4801 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
     4861 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
     4921 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
     4981 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
     5041 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
     5101 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
     5161 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat
     5221 tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc
     5281 gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag
     5341 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
     5401 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
     5461 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
     5521 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
     5581 atcgagatct cgatcccgcg aaat
//
