LOCUS       sequence-274646-        4172 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Provides IRES-puro-pA cassette on Gateway entry vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4172)
  TITLE     Minoru Ko Lab plasmids
REFERENCE   2  (bases 1 to 4172)
  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-IRES-puro-pA
FEATURES             Location/Qualifiers
     source          1..4172
                     /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    join(763..1337,1338..1340,1341..1349)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(930..947)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1157..1176
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             1350..1949
                     /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(1350..1369)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1846..1866
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(2030..2047)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2036..2260
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(2281..2376)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2413..2432)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2414..2432)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2437..2453)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2437..2453)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2555..2574)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2566..3375
                     /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(2633..2652)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3522..4110
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4011..4030
                     /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 cctccccccc
      781 ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt
      841 attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt
      901 cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa
      961 tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac
     1021 cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg
     1081 tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt
     1141 tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca
     1201 gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tgcacatgct ttacatgtgt
     1261 ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg
     1321 aaaaacacga tgataatatg gccacaacca tgaccgagta caagcccacg gtgcgcctcg
     1381 ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc
     1441 ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag
     1501 aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg
     1561 ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga
     1621 tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag
     1681 gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct
     1741 cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg
     1801 ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct
     1861 acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct
     1921 ggtgcatgac ccgcaagccc ggtgcctgac gcccgcccca cgacccgcag cgcccgaccg
     1981 aaaggagcgc acgaccccat gcatcgatga tctagagctc gctgatcagc ctcgactgtg
     2041 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     2101 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     2161 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     2221 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
     2281 caactttatt atacaaagtt ggcattataa aaaagcattg cttatcaatt tgttgcaacg
     2341 aacaggtcac tatcagtcaa aataaaatca ttatttggag ctccatggta gcgttaacgc
     2401 ggccgcgata tcccctatag tgagtcgtat tacatggtca tagctgtttc ctggcagctc
     2461 tggcccgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatga
     2521 acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa
     2581 cgggaaacgt cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa
     2641 tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta tgggaagccc
     2701 gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat
     2761 gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt
     2821 atccgtactc ctgatgatgc atggttactc accactgcga tccccggaaa aacagcattc
     2881 caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc
     2941 ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt
     3001 cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat
     3061 gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca
     3121 ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac
     3181 gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag
     3241 gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt
     3301 tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc
     3361 gatgagtttt tctaatcaga attggttaat tggttgtaac actggcagag cattacgctg
     3421 acttgacggg acggcgcaag ctcatgacca aaatccctta acgtgagtta cgcgtcgttc
     3481 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     3541 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     3601 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     3661 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     3721 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     3781 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     3841 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     3901 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     3961 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     4021 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     4081 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     4141 ctggcctttt gctggccttt tgctcacatg tt
//
