LOCUS       Exported                7488 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses SH1446 in E. coli with a C-terminal TEV-GFP-His(8) tag..
ACCESSION   .
VERSION     .
KEYWORDS    pWaldo-GFPe-SH1446
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7488)
  AUTHORS   Minhas GS, Bawdon D, Herman R, Rudden M, Stone AP, James AG, Thomas 
            GH, Newstead S
  TITLE     Structural basis of malodour precursor transport in the human 
            axilla.
  JOURNAL   Elife. 2018 Jul 3;7. pii: 34995. doi: 10.7554/eLife.34995.
  PUBMED    29966586
REFERENCE   2  (bases 1 to 7488)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7488
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     106..125
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     209..228
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        209..227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    228..252
                     /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             267..289
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     regulatory      853..862
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1805..1825
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1832..2542
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKAN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(1859..1887)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2482..2502
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             2555..2578
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(2631..2649)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2645..2692
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2729..3184
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2816..2835)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3026..3047
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3276..4091)
                     /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     3999..4018
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4083..4102
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4213..4801
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4702..4721
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4955..4972
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4987..5126
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5052..5071)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5212..5234
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5228..5419)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6228..7310)
                     /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     7272..7291
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7311..7388)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagac tcgacatggc
      301 aacaaataac tcccatgaac aaacaattca atcaatacca cagaaaggat ttttcggtca
      361 tcctagggga ctaggtgtgc tctttttcgt agagttttgg gaaagattca gttactacgg
      421 tatgcgtgcg atgctcatat tttatatgta tttcgcaata caccagaatg gattaggtat
      481 tgataaaact acagcaatgt caattatgtc agtatatgga gcactgattt acatgtcttc
      541 tattcctggc gcatggattg ctgatagaat tacaggcaca cgtggtgcta ctttattagg
      601 tgctgtactt attattattg gacatatctg tttaagttta ccattcgcct tatttggttt
      661 attctcatca atgttcttta ttattatcgg atcaggttta atgaaaccta atatttcaaa
      721 tattgtaggt cgtttatatc ctgaaaatga tacacgtatt gatgcaggtt tcgttatttt
      781 ctatatgtct gttaacttag gtgcattaat ttcacctatc atactgcaac attttgtaga
      841 tatacgtaat ttccatggtg gcttccttct tgcagctatt ggtatggcat taggtcttgt
      901 ttggtattta ttatttaata gaaaaaattt aggtagtgtt ggaatgaaac ctacgaatcc
      961 actatctaaa gaagaaaagc gtaaatatgg aatgattatc ggtatcatag tagcaattgt
     1021 gattgtcgta ttattagtta catattatac ccatacactt tccttcgatt taattagtaa
     1081 tactgtatta gttctaggag tagctttacc aatcatttac tttacaacta tgcttcgtag
     1141 taaagacgtt accgatggag aacgttctcg agtaaaagca tttatacctt tatttatttt
     1201 aggtatgtta ttctggtcaa ttcaagaaca aggttctaac gttttaaata tttatggttt
     1261 ggaacgttct gacatgcaac tcaacttatt tggttggaca actcgatttg gagaagcttt
     1321 gttccaatct atcaatccat tatttatttt attatttgca ccagttattt ctatgatttg
     1381 gttaaaaatg ggtaaaaagc aaccttcttt agctattaaa tttagtattg gtacattact
     1441 agcaggttta tcatacattc taattggatt agttggttta ggttatggac atacacaatt
     1501 ctcagttaac tgggttattt tatcatatgt aatctgtgtt attggggaac tttgtttatc
     1561 acctacaggt aatagtgctg ctgttaaact tgcacctaaa gcatttaatg ctcaaatgat
     1621 gagtgtatgg ctattaacaa atgcttcagc gcaagcaatt aacggtacat tagtaaaatt
     1681 aattaaacca cttggtcaaa ctaattattt tattttctta ggcacagttg ctattgttat
     1741 tactcttatc attttagttt tctcacctaa gattacgaaa gctatgaaag gtattcacgg
     1801 atccgaaaac ctgtacttcc agggtcaatt cagcaaagga gaagaacttt tcactggagt
     1861 tgtcccaatt cttgttgaat tagatggtga tgttaatggg cacaaatttt ctgtcagtgg
     1921 agagggtgaa ggtgatgcta catacggaaa actcaccctt aaatttattt gcactactgg
     1981 aaaactacct gttccatggc caacacttgt cactactctg acctatggtg ttcaatgctt
     2041 ttcccgttat ccggatcaca tgaaacggca tgactttttc aagagtgcca tgcccgaagg
     2101 ttatgtacag gaacgcacta tatctttcaa agatgacggg aactacaaga cgcgtgctga
     2161 agtcaagttt gaaggtgata cccttgttaa tcgtatcgag ttaaagggta ttgattttaa
     2221 agaagatgga aacattctcg gacacaaact agagtacaac tataactcac acaatgtata
     2281 catcacggca gacaaacaaa agaatggaat caaagctaac ttcaaaattc gccacaacat
     2341 tgaagatggt tccgttcaac tagcagacca ttatcaacaa aatactccaa ttggcgatgg
     2401 ccctgtcctt ttaccagaca accattacct gtcgacacaa tctgcccttt cgaaagatcc
     2461 caacgaaaag cgtgaccaca tggtccttct tgagtttgta actgctgctg ggattacaca
     2521 tggcatggat gagctctaca aaaagcttgc ggcccatcat catcaccacc accaccactg
     2581 agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
     2641 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
     2701 aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcacggcg
     2761 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     2821 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     2881 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     2941 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     3001 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     3061 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     3121 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     3181 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     3241 tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa
     3301 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
     3361 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
     3421 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
     3481 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt
     3541 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
     3601 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga
     3661 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
     3721 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
     3781 ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
     3841 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
     3901 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
     3961 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
     4021 tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga
     4081 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat
     4141 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     4201 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     4261 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     4321 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     4381 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     4441 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     4501 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     4561 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     4621 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     4681 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     4741 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     4801 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     4861 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     4921 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     4981 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg
     5041 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     5101 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     5161 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     5221 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     5281 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     5341 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     5401 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     5461 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
     5521 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
     5581 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
     5641 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
     5701 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
     5761 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
     5821 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
     5881 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
     5941 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
     6001 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
     6061 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
     6121 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
     6181 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
     6241 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     6301 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc
     6361 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc
     6421 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct
     6481 tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta
     6541 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg
     6601 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct
     6661 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga
     6721 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc
     6781 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg
     6841 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct
     6901 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt
     6961 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc
     7021 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc
     7081 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc
     7141 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact
     7201 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga
     7261 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc
     7321 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg
     7381 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag
     7441 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatg
//
