LOCUS Exported 5409 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Used as a PCR template for tagging yeast proteins with the DHFR tag (c-terminal histag) by homologous recombination and selection for hygromycin resistance.. ACCESSION . VERSION . KEYWORDS pAAH1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5409) AUTHORS Hoskins AA, Friedman LJ, Gallagher SS, Crawford DJ, Anderson EG, Wombacher R, Ramirez N, Cornish VW, Gelles J, Moore MJ TITLE Ordered and dynamic assembly of single spliceosomes. JOURNAL Science. 2011 Mar 11;331(6022):1289-95. PUBMED 21393538 REFERENCE 2 (bases 1 to 5409) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5409 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 23..42 /label=T7 /note="T7 promoter, forward primer" promoter 23..41 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" RBS 73..95 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 103..579 /codon_start=1 /gene="folA" /product="E. coli dihydrofolate reductase" /label=ecDHFR /translation="MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRH TWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFL PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR" CDS 580..597 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" gene 715..2290 /label=hphMX6 /note="yeast selectable marker conferring hygromycin resistance" promoter 715..1058 /label=TEF promoter /note="Ashbya gossypii TEF promoter" CDS 1059..2087 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA KE" terminator 2093..2290 /label=TEF terminator /note="Ashbya gossypii TEF terminator" primer_bind complement(2406..2424) /label=T7 Term /note="T7 terminator, reverse primer" terminator 2420..2467 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" primer_bind complement(2718..2735) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2889..3477) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2969..2988) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3648..4508) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 4271..4290 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4509..4613) /gene="bla" /label=AmpR promoter primer_bind 4681..4699 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(4737..4759) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 4859..4878 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" rep_origin complement(4896..5409) /direction=LEFT /label=M13 ori /note="M13 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5091..5112) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 5303..5322 /label=F1ori-R /note="F1 origin, reverse primer" ORIGIN 1 ggggatctcg atcccgcgaa attaatacga ctcactatag ggagaccaca acggtttccc 61 tctagaaata attttgttta actttaagaa ggagatatac atatgatcag tctgattgcg 121 gcgttagcgg tagatcgcgt tatcggcatg gaaaacgcca tgccgtggaa cctgcctgcc 181 gatctcgcct ggtttaaacg caacacctta aataaacccg tgattatggg ccgccatacc 241 tgggaatcaa tcggtcgtcc gttgccagga cgcaaaaata ttatcctcag cagtcaaccg 301 ggtacggacg atcgcgtaac gtgggtgaag tcggtggatg aagccatcgc ggcgtgtggt 361 gacgtaccag aaatcatggt gattggcggc ggtcgcgttt atgaacagtt cttgccaaaa 421 gcgcaaaaac tgtatctgac gcatatcgac gcagaagtgg aaggcgacac ccatttcccg 481 gattacgagc cggatgactg ggaatcggta ttcagcgaat tccacgatgc tgatgcgcag 541 aactctcaca gctattgctt tgagattctg gagcggcggc atcatcatca tcatcattaa 601 ctcgagcagc tgaagcttgc atgcctgcag gtcgactcta gaggatcccc gggttaatta 661 aggcgcgcca gatctgttta gcttgcctcg tccccgccgg gtcacccggc cagcgacatg 721 gaggcccaga ataccctcct tgacagtctt gacgtgcgca gctcaggggc atgatgtgac 781 tgtcgcccgt acatttagcc catacatccc catgtataat catttgcatc catacatttt 841 gatggccgca cggcgcgaag caaaaattac ggctcctcgc tgcagacctg cgagcaggga 901 aacgctcccc tcacagacgc gttgaattgt ccccacgccg cgcccctgta gagaaatata 961 aaaggttagg atttgccact gaggttcttc tttcatatac ttccttttaa aatcttgcta 1021 ggatacagtt ctcacatcac atccgaacat aaacaaccat gggtaaaaag cctgaactca 1081 ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc gacctgatgc 1141 agctctcgga gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg 1201 tcctgcgggt aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact 1261 ttgcatcggc cgcgctcccg attccggaag tgcttgacat tggggaattc agcgagagcc 1321 tgacctattg catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg 1381 aactgcccgc tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc 1441 ttagccagac gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat 1501 ggcgtgattt catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg 1561 acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg 1621 actgccccga agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg 1681 acaatggccg cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat 1741 acgaggtcgc caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc 1801 gctacttcga gcggaggcat ccggagcttg caggatcgcc gcggctccgg gcgtatatgc 1861 tccgcattgg tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag 1921 cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta 1981 cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg 2041 atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa ggaataatca gtactgacaa 2101 taaaaagatt cttgttttca agaacttgtc atttgtatag tttttttata ttgtagttgt 2161 tctattttaa tcaaatgtta gcgtgattta tatttttttt cgcctcgaca tcatctgccc 2221 agatgcgaag ttaagtgcgc agaaagtaat atcatgcgtc aatcgtatgt gaatgctggt 2281 cgctatactg ctgtcgattc gatactaacg ccgccatcca gtgtcgaaaa cgagctcgaa 2341 ttcatcgatg atatcagatc cggctgctaa caaagcccga aaggaagctg agttggctgc 2401 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg 2461 ttttttgctg aaaggaggaa ctatatccgg atcggatatc acaggacggg tgtggtcgcc 2521 atgatcgcgt agtcgatagt ggctccaagt agcgaagcga gcaggactgg gcggcggcca 2581 aagcggtcgg acagtgctcc gagaacgggt gcgcatagaa attgcatcaa cgcatatagc 2641 gctagagtcg acctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 2701 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 2761 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 2821 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 2881 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 2941 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 3001 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 3061 tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc 3121 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 3181 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 3241 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 3301 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag 3361 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 3421 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 3481 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 3541 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 3601 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 3661 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc 3721 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 3781 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 3841 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 3901 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 3961 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 4021 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 4081 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 4141 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 4201 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 4261 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 4321 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 4381 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 4441 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 4501 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 4561 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 4621 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa 4681 aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc 4741 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga 4801 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg 4861 gcatcagagc agattgtact gagagtgcag tacccacccc ggttgataat cagaaaagcc 4921 ccaaaaacag gaagattgta taagcaaata tttaaattgt aaacgttaat attttgttaa 4981 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca 5041 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga 5101 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc 5161 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc 5221 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc 5281 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg 5341 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac 5401 agggcgcgt //