LOCUS       sequence-274676-        4235 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Provides HA 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 4235)
  TITLE     Minoru Ko Lab plasmids
REFERENCE   2  (bases 1 to 4235)
  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-cHA-IRESpuro-pA
FEATURES             Location/Qualifiers
     source          1..4235
                     /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"
     CDS             756..782
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    join(825..1399,1400..1402,1403..1411)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(992..1009)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1219..1238
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             1412..2011
                     /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(1412..1431)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1908..1928
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(2092..2109)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2098..2323
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(2344..2439)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2476..2495)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2477..2495)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2500..2516)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2500..2516)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2618..2637)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2629..3438
                     /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(2696..2715)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3585..4173
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4074..4093
                     /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 tggcctaccc atacgacgtg ccagactacg
      781 cctagcggcc gctcgataag cttgatatcg aattccgccc cccccccctc tccctccccc
      841 ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg
      901 ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc tggccctgtc
      961 ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca aggtctgttg
     1021 aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg
     1081 accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca
     1141 cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt gagttggata
     1201 gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct gaaggatgcc
     1261 cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg ctttacatgt
     1321 gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt ggttttcctt
     1381 tgaaaaacac gatgataata tggccacaac catgaccgag tacaagccca cggtgcgcct
     1441 cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta
     1501 ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca
     1561 agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg
     1621 cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga
     1681 gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga
     1741 aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt
     1801 ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc
     1861 ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt
     1921 ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac
     1981 ctggtgcatg acccgcaagc ccggtgcctg acgcccgccc cacgacccgc agcgcccgac
     2041 cgaaaggagc gcacgacccc atgcatcgat gatctagagc tcgctgatca gcctcgactg
     2101 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     2161 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     2221 gtaggtgtca ttctattctg ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     2281 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     2341 acccaacttt attatacaaa gttggcatta taaaaaagca ttgcttatca atttgttgca
     2401 acgaacaggt cactatcagt caaaataaaa tcattatttg gagctccatg gtagcgttaa
     2461 cgcggccgcg atatccccta tagtgagtcg tattacatgg tcatagctgt ttcctggcag
     2521 ctctggcccg tgtctcaaaa tctctgatgt tacattgcac aagataaaaa tatatcatca
     2581 tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt
     2641 caacgggaaa cgtcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat
     2701 aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgctt gtatgggaag
     2761 cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca
     2821 gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat
     2881 tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg aaaaacagca
     2941 ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg
     3001 ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta
     3061 tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt
     3121 gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca taaacttttg
     3181 ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt
     3241 gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac
     3301 caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg
     3361 ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg
     3421 ctcgatgagt ttttctaatc agaattggtt aattggttgt aacactggca gagcattacg
     3481 ctgacttgac gggacggcgc aagctcatga ccaaaatccc ttaacgtgag ttacgcgtcg
     3541 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     3601 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     3661 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     3721 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     3781 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     3841 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     3901 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     3961 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     4021 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     4081 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     4141 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     4201 ttcctggcct tttgctggcc ttttgctcac atgtt
//
