LOCUS       sequence-274620-        5000 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Provides GR protein tag N-terminal to IRES-puro-pA cassette on 
            Gateway entry vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5000)
  TITLE     Minoru Ko Lab plasmids
REFERENCE   2  (bases 1 to 5000)
  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-cGR-IRESpuro-pA
FEATURES             Location/Qualifiers
     source          1..5000
                     /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(1591..2165,2166..2168,2169..2177)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1758..1775)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1985..2004
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2178..2777
                     /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(2178..2197)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2674..2694
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(2858..2875)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2864..3088
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(3109..3204)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3241..3260)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3242..3260)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3265..3281)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3265..3281)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3383..3402)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3394..4203
                     /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(3461..3480)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4350..4938
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4839..4858
                     /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 aaagtggcca tggaaaatcc tggtaacaaa acaatagttc
      781 ctgcaacgtt accacaactc acccctaccc tggtgtcact gttggaggtt attgaacctg
      841 aagtgttata tgcaggatat gatagctctg ttccagactc aacttggagg atcatgacta
      901 cgctcaacat gttaggaggg cggcaagtga ttgcagcagt gaaatgggca aaggcaatac
      961 caggtttcag gaacttacac ctggatgacc aaatgaccct actgcagtac tcctggatgt
     1021 ttcttatggc atttgctctg gggtggagat catatagaca atcaagtgca aacctgctgt
     1081 gttttgctcc tgatctgatt attaatgagc agagaatgac tctaccctgc atgtacgacc
     1141 aatgtaaaca catgctgtat gtttcctctg agttacacag gcttcaggta tcttatgaag
     1201 agtatctctg tatgaaaacc ttactgcttc tctcttcagt tcctaaggac ggtctgaaga
     1261 gccaagagct atttgatgaa attagaatga cctacatcaa agagctagga aaagccattg
     1321 tcaagaggga aggaaactcc agccagaact ggcagcggtt ttatcaactg acaaaactct
     1381 tggattctat gcatgaagtg gttgaaaatc tccttaacta ttgcttccaa acatttttgg
     1441 ataagaccat gtccatcgag ttccccgaga tgttagctga aatcatcacc aatcagatac
     1501 caaaatattc aaatggaaat atcaaaaaac ttctgtttca tcaaaagtga gcggccgctc
     1561 gataagcttg atatcgaatt ccgccccccc cccctctccc tccccccccc ctaacgttac
     1621 tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc tatatgttat tttccaccat
     1681 attgccgtct tttggcaatg tgagggcccg gaaacctggc cctgtcttct tgacgagcat
     1741 tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga
     1801 agcagttcct ctggaagctt cttgaagaca aacaacgtct gtagcgaccc tttgcaggca
     1861 gcggaacccc ccacctggcg acaggtgcct ctgcggccaa aagccacgtg tataagatac
     1921 acctgcaaag gcggcacaac cccagtgcca cgttgtgagt tggatagttg tggaaagagt
     1981 caaatggctc tcctcaagcg tattcaacaa ggggctgaag gatgcccaga aggtacccca
     2041 ttgtatggga tctgatctgg ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt
     2101 aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg
     2161 ataatatggc cacaaccatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg
     2221 acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc
     2281 acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca
     2341 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg
     2401 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca
     2461 tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc
     2521 cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc
     2581 agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg
     2641 gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg
     2701 gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc
     2761 gcaagcccgg tgcctgacgc ccgccccacg acccgcagcg cccgaccgaa aggagcgcac
     2821 gaccccatgc atcgatgatc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc
     2881 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     2941 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     3001 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     3061 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccca actttattat
     3121 acaaagttgg cattataaaa aagcattgct tatcaatttg ttgcaacgaa caggtcacta
     3181 tcagtcaaaa taaaatcatt atttggagct ccatggtagc gttaacgcgg ccgcgatatc
     3241 ccctatagtg agtcgtatta catggtcata gctgtttcct ggcagctctg gcccgtgtct
     3301 caaaatctct gatgttacat tgcacaagat aaaaatatat catcatgaac aataaaactg
     3361 tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtcg
     3421 aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat
     3481 aatgtcgggc aatcaggtgc gacaatctat cgcttgtatg ggaagcccga tgcgccagag
     3541 ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga
     3601 ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct
     3661 gatgatgcat ggttactcac cactgcgatc cccggaaaaa cagcattcca ggtattagaa
     3721 gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg
     3781 cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag
     3841 gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat
     3901 ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat
     3961 tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta
     4021 ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc
     4081 ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat
     4141 ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc
     4201 taatcagaat tggttaattg gttgtaacac tggcagagca ttacgctgac ttgacgggac
     4261 ggcgcaagct catgaccaaa atcccttaac gtgagttacg cgtcgttcca ctgagcgtca
     4321 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     4381 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     4441 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     4501 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     4561 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     4621 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     4681 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     4741 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     4801 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     4861 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     4921 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     4981 tggccttttg ctcacatgtt
//
