LOCUS       sequence-358191-        3243 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template plasmid for PCR amplification of initial recombineering 
            cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3243)
  AUTHORS   Nam HS, Capecchi MR
  TITLE     Additional gene targeting vector construction plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3243)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pMOD4-mT2A-mutated_rpsL-BSD-F
FEATURES             Location/Qualifiers
     source          1..3243
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     433..450
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    516..534
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     CDS             620..673
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1212..1610
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     protein_bind    complement(1649..1682)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     misc_feature    1853..1871
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     primer_bind     complement(2010..2029)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2129..2151
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2189..2207)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2275..2379
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2380..2448,2449..3240)
                     /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     complement(2598..2617)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttgtg
       61 catccgaaga tcagcagttc aacctgttga tagtacgtac taagctctca tgtttcacgt
      121 actaagctct catgtttaac gtactaagct ctcatgttta acgaactaaa ccctcatggc
      181 taacgtacta agctctcatg gctaacgtac taagctctca tgtttgaaca ataaaattaa
      241 tataaatcag caacttaaat agcctctaag gttttaagtt ttataagaaa aaaaagaata
      301 tataaggctt ttaaagctag cttttaaggt ttcaccatgt tctttcctgc gttatcccct
      361 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
      421 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
      481 cctctccccg cgcgttggcc gattcattaa gacagctgtc tcttatacac atctcaaccc
      541 tgaagctctt gttggctagt gcgtagtcgt tggcaagctt gcatgcctgc aggtcgacgg
      601 aggatctggc ggaagatctg aaggccgcgg cagcctgctg acatgcggcg acgtggaaga
      661 gaaccctggc cctgctcctg gcagcacagg cctggtgatg atggcgggat cgttgtatat
      721 ttcttgacac cttttcggca tcgccctaaa attcggcgtc ctcatattgt gtgaggacgt
      781 tttattacgt gtttacgaag caaaagctaa aaccaggagc tatttaatgg caacagttaa
      841 ccagctggta cgcaaaccac gtgctcgcaa agttgcgaaa agcaacgtgc ctgcgctgga
      901 agcatgcccg caaaacgtgg cgtatgtact cgtgtatata ctaccactcc taaaaaaccg
      961 aactccgcgc tgcgtaaagt atgccgtgtt cgtctgacta acggtttcga agtgacttcc
     1021 tacatcggtg gtgaaggtca caacctgcag gagcactccg tgatcctgat ccgtggcggt
     1081 cgtgttaaag acctcccggg tgttcgttac cacaccgtac gtggtgcgct tgactgctcc
     1141 ggcgttaaag accgtaagca ggctcgttcc aagtatggcg tgaagcgtcc taaggcttaa
     1201 ggaggacaat catggccaag cctttgtctc aagaagaatc caccctcatt gaaagagcaa
     1261 cggctacaat caacagcatc cccatctctg aagactacag cgtcgccagc gcagctctct
     1321 ctagcgacgg ccgcatcttc actggtgtca atgtatatca ttttactggg ggaccttgtg
     1381 cagaactcgt ggtgctgggc actgctgctg ctgcggcagc tggcaacctg acttgtatcg
     1441 tcgcgatcgg aaatgagaac aggggcatct tgagcccctg cggacggtgc cgacaggtgc
     1501 ttctcgatct gcatcctggg atcaaagcca tagtgaagga cagtgatgga cagccgacgg
     1561 cagttgggat tcgtgaattg ctgccctctg gttatgtgtg ggagggctaa aagctagctt
     1621 ggctggacgt aaactcctct tcagacctga agttcctata ctttctagag aataggaact
     1681 tcggaattcg atatcaagct tatcgatacc ggatcccttc ttgagtttgt aacagctgct
     1741 gggattacac atggcatgga cgaactatac aaaagcccac aagctttggg taccgagctc
     1801 gaattcgagc caatatgcga gaacacccga gaaaattcat cgatgatggt tgagatgtgt
     1861 ataagagaca gctgtcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     1921 cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     1981 atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     2041 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     2101 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     2161 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     2221 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     2281 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     2341 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     2401 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     2461 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     2521 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     2581 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     2641 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     2701 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     2761 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     2821 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     2881 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     2941 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     3001 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     3061 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     3121 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     3181 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     3241 ctg
//
