LOCUS       sequence-360644-        4297 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of DARPinOFF7.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4297)
  AUTHORS   Eastwood TA, Baker K, Streather BR, Allen N, Wang L, Botchway SW, 
            Brown IR, Hiscock JR, Lennon C, Mulvihill DP
  TITLE     High-yield vesicle-packaged recombinant protein production from E. 
            coli.
  JOURNAL   Cell Rep Methods. 2023 Feb 2;3(2):100396. doi: 
            10.1016/j.crmeth.2023.100396. eCollection 2023 Feb 27.
  PUBMED    36936078
REFERENCE   2  (bases 1 to 4297)
  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"; journalName: "Cell Rep 
            Methods."; date: "2023-02-2"; volume: "3(2):100396. doi"; pages: " 
            10.1016/j.crmeth.2023.100396. eCollection 2023 Feb 27"
COMMENT     Sequence Label: pRSFDuet-1_DARPinOFF7
FEATURES             Location/Qualifiers
     source          1..4297
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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)."
     CDS             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        214..232
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    233..257
                     /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)."
     CDS             801..818
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             834..878
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(916..934)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      930..977
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1207..2022)
                     /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(1207..2022)
                     /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     1930..1949
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2023..2114)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2130..2879)
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     complement(2140..2157)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2380..2399)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3075..4157)
                     /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     4119..4138
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4158..4235)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(4281..4297,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cgaattcgag
      121 ctcggcgcgc ctgcaggtcg acaagcttgc ggccgcataa tgcttaagtc gaacagaaag
      181 taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt
      241 gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatatacata
      301 tgggaggaag cggaatgggt tcggacttag gccgcaagtt actggaagct gcacgcgcgg
      361 gtcaagacga cgaggtacgc atccttatgg ccaatggtgc ggacgtcaac gccgccgata
      421 ataccggaac aactcctttg cacctcgcag catattccgg ccatttagag attgtagagg
      481 tactgcttaa gcacggtgcc gatgtagatg cgtcggacgt attcgggtac acgcctctcc
      541 acctcgccgc ttattggggc cacttagaaa ttgtagaggt gctcttgaag aatggtgccg
      601 acgtgaatgc tatggattca gacggcatga caccgctcca tctggccgcg aaatggggtt
      661 atctggagat cgtcgaggtt ctcttgaaac atggcgccga cgtcaatgca caagataagt
      721 tcggcaagac tgccttcgac atcagcatcg acaacggtaa tgaggacctg gccgagatcc
      781 ttcaaaagtt gaatggtacc catcatcatc atcatcatta actcgagtct ggtaaagaaa
      841 ccgctgctgc gaaatttgaa cgccagcaca tggactcgtc tactagcgca gcttaattaa
      901 cctaggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
      961 tcttgagggg ttttttgctg aaacctcagg catttgagaa gcacacggtc acactgcttc
     1021 cggtagtcaa taaaccggta aaccagcaat agacataagc ggctatttaa cgaccctgcc
     1081 ctgaaccgac gacaagctga cgaccgggtc tccgcaagtg gcacttttcg gggaaatgtg
     1141 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat
     1201 taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
     1261 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
     1321 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
     1381 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
     1441 gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac
     1501 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
     1561 tgattgcgcc tgagcgagac gaaatacgcg gtcgctgtta aaaggacaat tacaaacagg
     1621 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
     1681 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca
     1741 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     1801 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     1861 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg
     1921 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     1981 tcgcggccta gagcaagacg tttcccgttg aatatggctc atactcttcc tttttcaata
     2041 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     2101 gaaaaataaa caaataggca tgcagcgctc ttccgcttcc tcgctcactg actcgctacg
     2161 ctcggtcgtt cgactgcggc gagcggtgtc agctcactca aaagcggtaa tacggttatc
     2221 cacagaatca ggggataaag ccggaaagaa catgtgagca aaaagcaaag caccggaaga
     2281 agccaacgcc gcaggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     2341 gacgctcaag ccagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     2401 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     2461 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgttgg tatctcagtt
     2521 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     2581 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     2641 cactggcagc agccattggt aactgattta gaggactttg tcttgaagtt atgcacctgt
     2701 taaggctaaa ctgaaagaac agattttggt gagtgcggtc ctccaaccca cttaccttgg
     2761 ttcaaagagt tggtagctca gcgaaccttg agaaaaccac cgttggtagc ggtggttttt
     2821 ctttatttat gagatgatga atcaatcggt ctatcaagtc aacgaacagc tattccgtta
     2881 ctctagattt cagtgcaatt tatctcttca aatgtagcac ctgaagtcag ccccatacga
     2941 tataagttgt aattctcatg ttagtcatgc cccgcgccca ccggaaggag ctgactgggt
     3001 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     3061 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     3121 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     3181 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     3241 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     3301 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc
     3361 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     3421 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     3481 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     3541 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     3601 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     3661 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     3721 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     3781 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     3841 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     3901 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     3961 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     4021 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     4081 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     4141 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     4201 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     4261 cgactcctgc attaggaaat taatacgact cactata
//
