LOCUS       sequence-274667-        5176 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Provides IRES-pacDeltaTK-pA cassette on Gateway entry vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5176)
  TITLE     Minoru Ko Lab plasmids
REFERENCE   2  (bases 1 to 5176)
  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"
COMMENT     Sequence Label: pENTR-R2L3-IRESpacDeltaTKpA
FEATURES             Location/Qualifiers
     source          1..5176
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(624..747)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     misc_feature    775..1342
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(936..953)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1163..1182
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             1356..1952
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1356..1375)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1852..1872
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1959..2954
                     /codon_start=1
                     /product="truncated herpes simplex virus thymidine kinase 
                     (Salomon et al., 1995)"
                     /label=Delta-TK
                     /note="confers sensitivity to nucleoside analogs such as 
                     acyclovir or ganciclovir"
                     /translation="MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWRV
                     LGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGS
                     SHAPPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGAL
                     PEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQCGGSWREDWGQLSGTAV
                     PPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRSMHVFI
                     LDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN"
     protein_bind    complement(3285..3380)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3417..3436)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3418..3436)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3441..3457)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3441..3457)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3559..3578)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3570..4379
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3637..3656)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4526..5114
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5015..5034
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggccctg cagctctaga gctcgaattc tacaggtcac
      601 taataccatc taagtagttg gttcatagtg actgcatatg ttgtgtttta cagtattatg
      661 tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt
      721 ctcgttcaac tttcttgtac aaagtgggaa ttccgccccc cccccctctc cctctccctc
      781 ccccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg cgtttgtcta
      841 tatgttattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga aacctggccc
      901 tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct
      961 gttgaatgtc gtgaaggaag cagttcctct ggaagcttct tgaagacaaa caacgtctgt
     1021 agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct gcggccaaaa
     1081 gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg
     1141 gatagttgtg gaaagagtca aatggctctc ctcaagcgta ttcaacaagg ggctgaagga
     1201 tgcccagaag gtaccccatt gtatgggatc tgatctgggg cctcggtgca catgctttac
     1261 atgtgtttag tcgaggttaa aaaaacgtct aggccccccg aaccacgggg acgtggtttt
     1321 cctttgaaaa acacgatgat aagcttgcca caaccatgac cgagtacaag cccacggtgc
     1381 gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg
     1441 actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc
     1501 tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg
     1561 acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg
     1621 ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga
     1681 tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg
     1741 gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg
     1801 aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc
     1861 ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc
     1921 gcacctggtg catgacccgc aagcccggtg ccggatccat gcccacgcta ctgcgggttt
     1981 atatagacgg tccccacggg atggggaaaa ccaccaccac gcaactgctg gtggccctgg
     2041 gttcgcgcga cgatatcgtc tacgtacccg agccgatgac ttactggcgg gtgctggggg
     2101 cttccgagac aatcgcgaac atctacacca cacaacaccg cctcgaccag ggtgagatat
     2161 cggccgggga cgcggcggtg gtaatgacaa gcgcccagat aacaatgggc atgccttatg
     2221 ccgtgaccga cgccgttctg gctcctcata tcggggggga ggctgggagc tcacatgccc
     2281 cgcccccggc cctcaccctc atcttcgacc gccatcccat cgccgccctc ctgtgctacc
     2341 cggccgcgcg gtaccttatg ggcagcatga ccccccaggc cgtgctggcg ttcgtggccc
     2401 tcatcccgcc gaccttgccc ggcaccaaca tcgtgcttgg ggcccttccg gaggacagac
     2461 acatcgaccg cctggccaaa cgccagcgcc ccggcgagcg gctggacctg gctatgctgg
     2521 ctgcgattcg ccgcgtttac gggctacttg ccaatacggt gcggtatctg cagtgcggcg
     2581 ggtcgtggcg ggaggactgg ggacagcttt cggggacggc cgtgccgccc cagggtgccg
     2641 agccccagag caacgcgggc ccacgacccc atatcgggga cacgttattt accctgtttc
     2701 gggcccccga gttgctggcc cccaacggcg acctgtataa cgtgtttgcc tgggccttgg
     2761 acgtcttggc caaacgcctc cgttccatgc acgtctttat cctggattac gaccaatcgc
     2821 ccgccggctg ccgggacgcc ctgctgcaac ttacctccgg gatggtccag acccacgtca
     2881 ccacccccgg ctccataccg acgatatgcg acctggcgcg cacgtttgcc cgggagatgg
     2941 gggaggctaa ctgagctcta gcttatcgac tcgccgatag tggaaaccga cgccccagca
     3001 ctcgtccgag ggcaaaggaa tagtcgagaa attgatgatc tattaaacaa taaagatgtc
     3061 cacatggaag tttttcctgt catactttgt taagaagggt gagaacagag tacctacatt
     3121 ttgaatggaa ggattggagc tacgggggtg ggggtggggt gggattagat aaatgcctgc
     3181 tctttactga aggctcttta ctattgcttt atgataatgt ttcatagttg gatatcataa
     3241 tttaaacaag caaaaccaaa ttaagggcca gctcacccac tcatcaactt tattatacaa
     3301 agttggcatt ataaaaaagc attgcttatc aatttgttgc aacgaacagg tcactatcag
     3361 tcaaaataaa atcattattt ggagctccat ggtagcgtta acgcggccgc gatatcccct
     3421 atagtgagtc gtattacatg gtcatagctg tttcctggca gctctggccc gtgtctcaaa
     3481 atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata aaactgtctg
     3541 cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcgaggc
     3601 cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg
     3661 tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg ccagagttgt
     3721 ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa
     3781 actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg
     3841 atgcatggtt actcaccact gcgatccccg gaaaaacagc attccaggta ttagaagaat
     3901 atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt
     3961 cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc
     4021 aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct
     4081 ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag
     4141 tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag
     4201 gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat
     4261 ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta
     4321 ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat
     4381 cagaattggt taattggttg taacactggc agagcattac gctgacttga cgggacggcg
     4441 caagctcatg accaaaatcc cttaacgtga gttacgcgtc gttccactga gcgtcagacc
     4501 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     4561 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     4621 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     4681 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     4741 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     4801 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     4861 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     4921 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     4981 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     5041 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     5101 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     5161 cttttgctca catgtt
//
