LOCUS       pMSP1D12W/2Y               5881 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Study of protein insertion into nanodisks by means of changes in 
            tryptophan fluorescence.
ACCESSION   .
VERSION     .
KEYWORDS    pMSP1D12W/2Y
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5881)
  TITLE     Bacterial expression plasmid for production of recombinant 
            MSP1D12W/2Y protein where 2 tryptophan residues are substituted with
            2 tyrosines
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5881)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            //www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5881
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     terminator      26..73
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(747..767)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(789..809)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     RBS             822..844
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    859..883
                     /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)."
     primer_bind     complement(883..902)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(884..902)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(986..1005)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1211..1288
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1289..2371
                     /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     complement(1308..1327)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3180..3371
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3365..3387)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3473..3612
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3528..3547
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3627..3644)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3798..4386)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3878..3897)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4497..4516)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4508..5323
                     /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     complement(4581..4600)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5415..5870)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5552..5573)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5764..5783
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttac
      181 tgggtattca gctttttagt atattcttcc agagctgaca gaaaactgac tttaaagctc
      241 tcaagaacag gcaataagcc ctggcgtaga tcttccagcg ccggtttcgc tttttcgctc
      301 aaggtgctca ggtgttctgt cgctttcgcg tggtactcag ccaagcgggc acccccgttt
      361 tctttcagcg cctcgagacg tgcggccaaa cgctggcgaa gttcatccga atacggcgcc
      421 aaatgagtcc ggagtgcatc aacatgtgca cgggcgcgat cgcgcatttc ttcgcctaat
      481 gggctgagct tctcttggag ctcatggagt ttttggcgtg ccccctcttg cagttccgca
      541 cgcagcggtt ccaccttttg acgatataat tccatctctt cctggtattt tttctgaaag
      601 tcatcgagat atggctgaac cttggccttc acctcttcta aatctttgga catttcctga
      661 cgcagtccct cggtttcttt ttccaggttg tcgtagaatt cctgcgtcac ggggcccagt
      721 tgttcgcgaa gtttactgaa ggtagaaccc tgaaaataca aattctcagt agtaggaata
      781 tcataatcgt gatgatgatg atgatgatga cccatggtat atctccttct taaagttaaa
      841 caaaattatt tctagagggg aattgttatc cgctcacaat tcccctatag tgagtcgtat
      901 taatttcgcg ggatcgagat ctcgatcctc tacgccggac gcatcgtggc cggcatcacc
      961 ggcgccacag gtgcggttgc tggcgcctat atcgccgaca tcaccgatgg ggaagatcgg
     1021 gctcgccact tcgggctcat gagcgcttgt ttcggcgtgg gtatggtggc aggccccgtg
     1081 gccgggggac tgttgggcgc catctccttg catgcaccat tccttgcggc ggcggtgctc
     1141 aacggcctca acctactact gggctgcttc ctaatgcagg agtcgcataa gggagagcgt
     1201 cgagatcccg gacaccatcg aatggcgcaa aacctttcgc ggtatggcat gatagcgccc
     1261 ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga
     1321 gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc
     1381 tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg
     1441 cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct
     1501 ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg
     1561 tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt
     1621 gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca
     1681 ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc
     1741 tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt
     1801 ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc
     1861 tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca
     1921 gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca
     1981 aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct
     2041 gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt
     2101 gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca
     2161 ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca
     2221 ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc
     2281 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     2341 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca
     2401 ctcattaggc accgggatct cgaccgatgc ccttgagagc cttcaaccca gtcagctcct
     2461 tccggtgggc gcggggcatg actatcgtcg ccgcacttat gactgtcttc tttatcatgc
     2521 aactcgtagg acaggtgccg gcagcgctct gggtcatttt cggcgaggac cgctttcgct
     2581 ggagcgcgac gatgatcggc ctgtcgcttg cggtattcgg aatcttgcac gccctcgctc
     2641 aagccttcgt cactggtccc gccaccaaac gtttcggcga gaagcaggcc attatcgccg
     2701 gcatggcggc cccacgggtg cgcatgatcg tgctcctgtc gttgaggacc cggctaggct
     2761 ggcggggttg ccttactggt tagcagaatg aatcaccgat acgcgagcga acgtgaagcg
     2821 actgctgctg caaaacgtct gcgacctgag caacaacatg aatggtcttc ggtttccgtg
     2881 tttcgtaaag tctggaaacg cggaagtcag cgccctgcac cattatgttc cggatctgca
     2941 tcgcaggatg ctgctggcta ccctgtggaa cacctacatc tgtattaacg aagcgctggc
     3001 attgaccctg agtgattttt ctctggtccc gccgcatcca taccgccagt tgtttaccct
     3061 cacaacgttc cagtaaccgg gcatgttcat catcagtaac ccgtatcgtg agcatcctct
     3121 ctcgtttcat cggtatcatt acccccatga acagaaatcc cccttacacg gaggcatcag
     3181 tgaccaaaca ggaaaaaacc gcccttaaca tggcccgctt tatcagaagc cagacattaa
     3241 cgcttctgga gaaactcaac gagctggacg cggatgaaca ggcagacatc tgtgaatcgc
     3301 ttcacgacca cgctgatgag ctttaccgca gctgcctcgc gcgtttcggt gatgacggtg
     3361 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
     3421 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca
     3481 tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca
     3541 gattgtactg agagtgcacc attgcggtgt gaaataccgc acagatgcgt aaggagaaaa
     3601 taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3661 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     3721 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     3781 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     3841 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     3901 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     3961 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4021 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4081 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4141 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     4201 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
     4261 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4321 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4381 tttcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4441 cgttaaggga ttttggtcat gaacaataaa actgtctgct tacataaaca gtaatacaag
     4501 gggtgttatg agccatattc aacgggaaac gtcttgctct aggccgcgat taaattccaa
     4561 catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc
     4621 gacaatctat cgattgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa
     4681 aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt
     4741 tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac
     4801 cactgcgatc cccgggaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga
     4861 aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa
     4921 ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa
     4981 cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt
     5041 ctggaaagaa atgcataaac ttttgccatt ctcaccggat tcagtcgtca ctcatggtga
     5101 tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg
     5161 acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga
     5221 gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat
     5281 gaataaattg cagtttcatt tgatgctcga tgagtttttc taagaattaa ttcatgagcg
     5341 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     5401 gaaaagtgcc acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     5461 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     5521 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     5581 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     5641 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     5701 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     5761 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     5821 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc
     5881 a
//