LOCUS       sequence-69168-e        4002 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used as a PCR template for tagging yeast proteins with the DHFR tag 
            (c-terminal histag) by homologous recombination and selection for 
            phleomycin resistance..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4002)
  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 4002)
  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: pAAH3
FEATURES             Location/Qualifiers
     source          1..4002
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             4..480
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRH
                     TWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFL
                     PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     CDS             481..498
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    548..581
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        636..979
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     terminator      1372..1569
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    1632..1665
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(1718..1737)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1719..1737)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1824..1841)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1995..2583)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2075..2094)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2754..3545,3546..3614))
                     /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     3377..3396
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3615..3719)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3787..3805
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3843..3865)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3965..3984
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 catatgatca gtctgattgc ggcgttagcg gtagatcgcg ttatcggcat ggaaaacgcc
       61 atgccgtgga acctgcctgc cgatctcgcc tggtttaaac gcaacacctt aaataaaccc
      121 gtgattatgg gccgccatac ctgggaatca atcggtcgtc cgttgccagg acgcaaaaat
      181 attatcctca gcagtcaacc gggtacggac gatcgcgtaa cgtgggtgaa gtcggtggat
      241 gaagccatcg cggcgtgtgg tgacgtacca gaaatcatgg tgattggcgg cggtcgcgtt
      301 tatgaacagt tcttgccaaa agcgcaaaaa ctgtatctga cgcatatcga cgcagaagtg
      361 gaaggcgaca cccatttccc ggattacgag ccggatgact gggaatcggt attcagcgaa
      421 ttccacgatg ctgatgcgca gaactctcac agctattgct ttgagattct ggagcggcgg
      481 catcatcatc atcatcatta actcgagcag ctgaagcttc gtacgctgca ggtcgacaac
      541 ccttaatata acttcgtata atgtatgcta tacgaagtta ttaggtctag agatctgttt
      601 agcttgcctc gtccccgccg ggtcacccgg ccagcgacat ggaggcccag aataccctcc
      661 ttgacagtct tgacgtgcgc agctcagggg catgatgtga ctgtcgcccg tacatttagc
      721 ccatacatcc ccatgtataa tcatttgcat ccatacattt tgatggccgc acggcgcgaa
      781 gcaaaaatta cggctcctcg ctgcagacct gcgagcaggg aaacgctccc ctcacagacg
      841 cgttgaattg tccccacgcc gcgcccctgt agagaaatat aaaaggttag gatttgccac
      901 tgaggttctt ctttcatata cttcctttta aaatcttgct aggatacagt tctcacatca
      961 catccgaaca taaacaacca tgggtatgac cgaccaagcg acgcccaacc tgccatcacg
     1021 agatttcgat cccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     1081 cgccggctgg atgatcctcc agcgcgggga tctcaagctg gagttcttcg cccaccccgg
     1141 gctcgatccc ctcgcgagtt ggttcagctg ctgcctgagg ctggacgacc tcgcggagtt
     1201 ctaccggcag tgcaaatccg tcggcatcca ggaaaccagc agcggctatc cgcgcatcca
     1261 tgcccccgaa ctgcaggagt ggggaggcac gatggccgct ttggtcgacc cggacgggac
     1321 gctcctgcgc ctgatacaga acgaattgct tgcaggcatc tcatgatcag tactgacaat
     1381 aaaaagattc ttgttttcaa gaacttgtca tttgtatagt ttttttatat tgtagttgtt
     1441 ctattttaat caaatgttag cgtgatttat attttttttc gcctcgacat catctgccca
     1501 gatgcgaagt taagtgcgca gaaagtaata tcatgcgtca atcgtatgtg aatgctggtc
     1561 gctatactgc tgtcgattcg atactaacgc cgccatccag tgtcgaaaac gagctctcga
     1621 gaacccttaa tataacttcg tataatgtat gctatacgaa gttattaggt gatatcagat
     1681 ccactagtgg cctatgcggc cgcggatctg ccggtctccc tatagtgagt cgtattaatt
     1741 tcgataagcc aggttaacct gcattaatga atcggccaac gcgcggggag aggcggtttg
     1801 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     1861 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     1921 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     1981 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     2041 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     2101 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     2161 ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg
     2221 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     2281 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     2341 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     2401 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     2461 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     2521 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     2581 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     2641 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     2701 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     2761 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     2821 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     2881 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     2941 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     3001 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     3061 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     3121 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     3181 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     3241 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     3301 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     3361 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     3421 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     3481 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     3541 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     3601 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     3661 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     3721 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
     3781 tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa
     3841 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     3901 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac
     3961 tatgcggcat cagagcagat tgtactgaga gtgcaccata tg
//
