LOCUS       sequence-274627-        6086 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Provides conditional promoter cassette and PGK promoter driven 
            puromycin resistant gene cassette between attL4-attR1 on Gateway 
            entry vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6086)
  TITLE     Minoru Ko Lab plasmids
REFERENCE   2  (bases 1 to 6086)
  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-L4R1-PHtetO2
FEATURES             Location/Qualifiers
     source          1..6086
                     /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     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    593..688
                     /gene="mutant version of attL"
                     /label=attL4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     743..759
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        770..1269
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(805..828)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1202..1221
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             1293..1889
                     /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(1293..1312)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1789..1809
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1896..2891
                     /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"
     primer_bind     complement(3525..3541)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(join(3573..3579,3580..3597,3598..3603))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3618..3639
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    3722..3740
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3759..3777
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3796..3814
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3833..3851
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3870..3888
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3907..3925
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3944..3962
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3981..3999
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     4021..4045
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    4132..4255
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4327..4346)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4328..4346)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4351..4367)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4469..4488)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4480..5289
                     /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(4547..4566)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      5436..6024
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5925..5944
                     /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 tcgggcccga gttaacgcta ccatggagct ccaaataatg
      601 attttatttt gactgatagt gacctgttcg ttgcaacaaa ttgataagca atgctttttt
      661 ataatgccaa ctttgtatag aaaagttgtt taaacgtcga cattaagttg ggtaacgcca
      721 gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattctaccg ggtaggggag
      781 gcgcttttcc caaggcagtc tggagcatgc gctttagcag ccccgctggg cacttggcgc
      841 tacacaagtg gcctctggcc tcgcacacat tccacatcca ccggtaggcg ccaaccggct
      901 ccgttctttg gtggcccctt cgcgccacct tctactcctc ccctagtcag gaagttcccc
      961 cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg tctcactagt
     1021 ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg taggcctttg gggcagcggc
     1081 caatagcagc tttgctcctt cgctttctgg gctcagaggc tgggaagggg tgggtccggg
     1141 ggcgggctca ggggcgggct caggggcggg gcgggcgccc gaaggtcctc cggaggcccg
     1201 gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc ctcatctccg
     1261 ggcctttcga ccgatcatca agcttgatcc tcatgaccga gtacaagccc acggtgcgcc
     1321 tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact
     1381 accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc
     1441 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg
     1501 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg
     1561 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg
     1621 aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg
     1681 tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg
     1741 cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct
     1801 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca
     1861 cctggtgcat gacccgcaag cccggtgccg gatccatgcc cacgctactg cgggtttata
     1921 tagacggtcc ccacgggatg gggaaaacca ccaccacgca actgctggtg gccctgggtt
     1981 cgcgcgacga tatcgtctac gtacccgagc cgatgactta ctggcgggtg ctgggggctt
     2041 ccgagacaat cgcgaacatc tacaccacac aacaccgcct cgaccagggt gagatatcgg
     2101 ccggggacgc ggcggtggta atgacaagcg cccagataac aatgggcatg ccttatgccg
     2161 tgaccgacgc cgttctggct cctcatatcg ggggggaggc tgggagctca catgccccgc
     2221 ccccggccct caccctcatc ttcgaccgcc atcccatcgc cgccctcctg tgctacccgg
     2281 ccgcgcggta ccttatgggc agcatgaccc cccaggccgt gctggcgttc gtggccctca
     2341 tcccgccgac cttgcccggc accaacatcg tgcttggggc ccttccggag gacagacaca
     2401 tcgaccgcct ggccaaacgc cagcgccccg gcgagcggct ggacctggct atgctggctg
     2461 cgattcgccg cgtttacggg ctacttgcca atacggtgcg gtatctgcag tgcggcgggt
     2521 cgtggcggga ggactgggga cagctttcgg ggacggccgt gccgccccag ggtgccgagc
     2581 cccagagcaa cgcgggccca cgaccccata tcggggacac gttatttacc ctgtttcggg
     2641 cccccgagtt gctggccccc aacggcgacc tgtataacgt gtttgcctgg gccttggacg
     2701 tcttggccaa acgcctccgt tccatgcacg tctttatcct ggattacgac caatcgcccg
     2761 ccggctgccg ggacgccctg ctgcaactta cctccgggat ggtccagacc cacgtcacca
     2821 cccccggctc cataccgacg atatgcgacc tggcgcgcac gtttgcccgg gagatggggg
     2881 aggctaactg agctctagct tatcgactcg ccgatagtgg aaaccgacgc cccagcactc
     2941 gtccgagggc aaaggaatag tcgagaaatt gatgatctat taaacaataa agatgtccac
     3001 atggaagttt ttcctgtcat actttgttaa gaagggtgag aacagagtac ctacattttg
     3061 aatggaagga ttggagctac gggggtgggg gtggggtggg attagataaa tgcctgctct
     3121 ttactgaagg ctctttacta ttgctttatg ataatgtttc atagttggat atcataattt
     3181 aaacaagcaa aaccaaatta agggccagct cattcctccc actcatgatc tatagatcct
     3241 ctagagagct cacggggaca gccccccccc aaagccccca gggatgtaat tacgtccctc
     3301 ccccgctagg gggcagcagc gagccgcccg gggctccgct ccggtccggc gctccccccg
     3361 catccccgag ccggcagcgt gcggggacag cccgggcacg gggaaggtgg cacgggatcg
     3421 ctttcctctg aacgcttctc gctgctcttt gagcctgcag acacctgggg gatacgggga
     3481 aaaagcttgt cgacctgcag gcatgcaagc ttggcgtaat catggtcata gctgtttcct
     3541 gtgtgaaatt gttatccgtt cacaattcca cacaacatac gagccggaag cataaagtgt
     3601 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     3661 gctttccagt cgggaaacct gtcgtgccag taactcgagg gatccaagct ggccgcacgt
     3721 ctccctatca gtgatagaga agtcgacacg tctcgagctc cctatcagtg atagagaagg
     3781 tacgtctaga acgtctccct atcagtgata gagaagtcga cacgtctcga gctccctatc
     3841 agtgatagag aaggtacgtc tagaacgtct ccctatcagt gatagagaag tcgacacgtc
     3901 tcgagctccc tatcagtgat agagaaggta cgtctagaac gtctccctat cagtgataga
     3961 gaagtcgaca cgtctcgagc tccctatcag tgatagagaa ggtaccccct atataagcag
     4021 agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca
     4081 tagaagacac cgggaccgat ccagcctccg cggtggcggc catcgcgatc gcaagtttgt
     4141 acaaaaaagt tgaacgagaa acgtaaaatg atataaatat caatatatta aattagattt
     4201 tgcataaaaa acagactaca taatactgta aaacacaaca tatgcagtca ctatgaacca
     4261 actacttaga tggtattagt gacctgtaga attcgagctc tagagctgca gggcggccgc
     4321 gatatcccct atagtgagtc gtattacatg gtcatagctg tttcctggca gctctggccc
     4381 gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata
     4441 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa
     4501 acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct
     4561 cgcgataatg tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg
     4621 ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg
     4681 gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt
     4741 actcctgatg atgcatggtt actcaccact gcgatccccg gaaaaacagc attccaggta
     4801 ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc
     4861 cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc
     4921 gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag
     4981 cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca
     5041 ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg
     5101 aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt
     5161 gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa
     5221 aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag
     5281 tttttctaat cagaattggt taattggttg taacactggc agagcattac gctgacttga
     5341 cgggacggcg caagctcatg accaaaatcc cttaacgtga gttacgcgtc gttccactga
     5401 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     5461 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     5521 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     5581 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     5641 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     5701 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     5761 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     5821 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     5881 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     5941 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     6001 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     6061 ttttgctggc cttttgctca catgtt
//
