LOCUS       sequence-98323-e        3760 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PCR template vector for generating methotrexate resistance 
            cassette..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3760)
  AUTHORS   MacDonald C, Piper RC
  TITLE     Puromycin and Methotrexate Resistance Cassettes and Optimized 
            cre-recombinase Expression Plasmids for use in Yeast.
  JOURNAL   Yeast. 2015 Feb 16. doi: 10.1002/yea.3069.
  PUBMED    25688547
REFERENCE   2  (bases 1 to 3760)
  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: "Yeast. 2015
            Feb 16. doi: 10.1002/yea.3069."
COMMENT     Sequence Label: pPL5618_pUG_MtxR
FEATURES             Location/Qualifiers
     source          1..3760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    53..86
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        141..484
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     terminator      1054..1251
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    1311..1344
                     /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(1397..1416)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1398..1416)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1503..1520)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1674..2262)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1754..1773)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2433..3224,3225..3293))
                     /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     3056..3075
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3294..3398)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3466..3484
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3522..3544)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3644..3663
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(3744..3760,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(3744..3760,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg ctgcaggtcg acaaccctta atataacttc
       61 gtataatgta tgctatacga agttattagg tctagagatc tgtttagctt gcctcgtccc
      121 cgccgggtca cccggccagc gacatggagg cccagaatac cctccttgac agtcttgacg
      181 tgcgcagctc aggggcatga tgtgactgtc gcccgtacat ttagcccata catccccatg
      241 tataatcatt tgcatccata cattttgatg gccgcacggc gcgaagcaaa aattacggct
      301 cctcgctgca gacctgcgag cagggaaacg ctcccctcac agacgcgttg aattgtcccc
      361 acgccgcgcc cctgtagaga aatataaaag gttaggattt gccactgagg ttcttctttc
      421 atatacttcc ttttaaaatc ttgctaggat acagttctca catcacatcc gaacataaac
      481 aaccatggtt ggttctttga actgtatcgt tgctgtttct caaaacatgg gtatcggtaa
      541 gaacggtgac ttcccatggc caccattgag aaacgaatct agatacttcc aaagaatgac
      601 caccacctct tctgttgaag gtaagcaaaa cttggttatc atgggtaaga agacctggtt
      661 ctctatccca gaaaagaaca gaccattgaa gggtagaatc aacttggttt tgtctagaga
      721 attgaaggaa ccaccacaag gtgctcactt cttgtctaga tctttggacg acgctttgaa
      781 gttgaccgaa caaccagaat tggctaacaa ggttgacatg gtttggatcg ttggtggttc
      841 ttctgtttac aaggaagcta tgaaccaccc aggtcacttg aagttgttcg ttaccagaat
      901 catgcaagac ttcgaatctg acaccttctt cccagaaatc gacttggaaa agtacaagtt
      961 gttgccagaa tacccaggtg ttttgtctga cgttcaagaa gaaaagggta tcaagtacaa
     1021 gttcgaagtt tacgaaaaga acgactaaag agtactgaca ataaaaagat tcttgttttc
     1081 aagaacttgt catttgtata gtttttttat attgtagttg ttctatttta atcaaatgtt
     1141 agcgtgattt atattttttt tcgcctcgac atcatctgcc cagatgcgaa gttaagtgcg
     1201 cagaaagtaa tatcatgcgt caatcgtatg tgaatgctgg tcgctatact gctgtcgatt
     1261 cgatactaac gccgccatcc agtttaaacg agctctcgag aacccttaat ataacttcgt
     1321 ataatgtatg ctatacgaag ttattaggtg atatcagatc cactagtggc ctatgcggcc
     1381 gcggatctgc cggtctccct atagtgagtc gtattaattt cgataagcca ggttaacctg
     1441 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     1501 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     1561 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     1621 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     1681 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     1741 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     1801 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     1861 ctttctcaat gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     1921 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     1981 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     2041 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     2101 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     2161 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     2221 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     2281 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     2341 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     2401 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     2461 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     2521 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     2581 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     2641 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     2701 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     2761 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     2821 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     2881 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     2941 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     3001 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     3061 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     3121 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     3181 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     3241 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     3301 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     3361 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     3421 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     3481 aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     3541 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     3601 gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt
     3661 gtactgagag tgcaccatat ggacatattg tcgttagaac gcggctacaa ttaatacata
     3721 accttatgta tcatacacat acgatttagg tgacactata
//
