LOCUS       sequence-69077-e        5409 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used as a PCR template for tagging yeast proteins with the DHFR tag 
            by homologous recombination and selection for hygromycin 
            resistance..
ACCESSION   .
VERSION     .
KEYWORDS    .
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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Science."; 
            date: "2011-03-11"; volume: "331(6022)"; pages: "1289-95"
COMMENT     Sequence Label: pAAH2
FEATURES             Location/Qualifiers
     source          1..5409
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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..582
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRH
                     TWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFL
                     PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     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
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     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(join(3648..4439,4440..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 gagcggcggt aacatcatca 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 tgcggacctg 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
//
