LOCUS       sequence-260259-        5853 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Bacterial expression vector for methyl-binding domain protein 2 
            (MBD2) coiled-coil domain for use as competitor peptide in 
            measurements of effective concentrations.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5853)
  AUTHORS   Sorensen CS, Kjaergaard M
  TITLE     Measuring Effective Concentrations Enforced by Intrinsically 
            Disordered Linkers.
  JOURNAL   Methods Mol Biol. 2020;2141:505-518. doi: 
            10.1007/978-1-0716-0524-0_25.
  PUBMED    32696374
REFERENCE   2  (bases 1 to 5853)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Methods Mol
            Biol."; date: "2020"; volume: "2141:505-518. doi"; pages: " 
            10.1007/978-1-0716-0524-0_25"
COMMENT     Sequence Label: Methyl-binding domain protein 2 (MBD2) coiled-...
FEATURES             Location/Qualifiers
     source          1..5853
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1..23
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             42..59
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             69..86
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(296..314)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      310..357
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(532..537,538..554,555..560))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(587..605)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        673..777
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(778..846,847..1638)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(996..1015)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1809..2397
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2298..2317
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2551..2568
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2583..2722
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2648..2667)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2808..2830
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2824..3015)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4327..5409)
                     /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     5371..5390
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5410..5487)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5693..5712
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5796..5815
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5796..5814
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5815..5839
                     /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)."
ORIGIN
        1 tttgtttaac tttaagaagg agatatacca tgggcagcag ccatcatcat catcatcaca
       61 gcagcggcct ggtgccgcgc ggcagccata tgcagagcca aagccaaagc caaagccaga
      121 gcgtgaccga cgaggatatc cgtaagcaag aggaacgtgt gcagcaagtt cgtaagaaac
      181 tggaggaagc gctgatggcg gatgcgagcg gtagcggcag cggtagcggc agcggtagct
      241 actaaggatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
      301 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
      361 aaaggaggaa ctatatccgg atatcccgca agaggcccgg cagtaccggc ataaccaagc
      421 ctatgcctac agcatccagg gtgacggtgc cgaggatgac gatgagcgca ttgttagatt
      481 tcatacacgg tgcctgactg cgttagcaat ttaactgtga taaactaccg cattaaagct
      541 tatcgatgat aagctgtcaa acatgagaat tcttgaagac gaaagggcct cgtgatacgc
      601 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
      661 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
      721 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
      781 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
      841 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
      901 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
      961 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     1021 gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     1081 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     1141 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     1201 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     1261 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     1321 gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     1381 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     1441 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     1501 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     1561 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     1621 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     1681 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     1741 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     1801 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     1861 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     1921 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     1981 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     2041 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     2101 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     2161 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     2221 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     2281 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     2341 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     2401 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     2461 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     2521 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     2581 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     2641 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     2701 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     2761 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     2821 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     2881 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     2941 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     3001 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     3061 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     3121 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     3181 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     3241 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     3301 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     3361 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     3421 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     3481 tggccaggac ccaacgctgc ccgagatgcg ccgcgtgcgg ctgctggaga tggcggacgc
     3541 gatggatatg ttctgccaag ggttggtttg cgcattcaca gttctccgca agaattgatt
     3601 ggctccaatt cttggagtgg tgaatccgtt agcgaggtgc cgccggcttc cattcaggtc
     3661 gaggtggccc ggctccatgc accgcgacgc aacgcgggga ggcagacaag gtatagggcg
     3721 gcgcctacaa tccatgccaa cccgttccat gtgctcgccg aggcggcata aatcgccgtg
     3781 acgatcagcg gtccaatgat cgaagttagg ctggtaagag ccgcgagcga tccttgaagc
     3841 tgtccctgat ggtcgtcatc tacctgcctg gacagcatgg cctgcaacgc gggcatcccg
     3901 atgccgccgg aagcgagaag aatcataatg gggaaggcca tccagcctcg cgtcgcgaac
     3961 gccagcaaga cgtagcccag cgcgtcggcc gccatgccgg cgataatggc ctgcttctcg
     4021 ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg
     4081 aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa
     4141 atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata
     4201 agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     4261 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     4321 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4381 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     4441 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     4501 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     4561 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac
     4621 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     4681 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     4741 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     4801 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     4861 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     4921 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     4981 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     5041 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     5101 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     5161 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     5221 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     5281 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     5341 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     5401 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     5461 aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct
     5521 gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg
     5581 gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca
     5641 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt
     5701 cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc
     5761 cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta taggggaatt
     5821 gtgagcggat aacaattccc ctctagaaat aat
//
