LOCUS       sequence-274617-        5229 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Provides pA sequences N-terminal to PGK-PacDeltaTK-pA cassette on 
            Gateway entry vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5229)
  TITLE     Minoru Ko Lab plasmids
REFERENCE   2  (bases 1 to 5229)
  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-pA-PGKpacDeltaTK-pA1
FEATURES             Location/Qualifiers
     source          1..5229
                     /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     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(624..748)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     polyA_signal    760..808
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     810..826
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        837..1336
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(872..895)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1269..1288
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             1360..1956
                     /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(1360..1379)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1856..1876
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1963..2958
                     /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(3338..3433)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3470..3489)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3471..3489)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3494..3510)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3494..3510)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3612..3631)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3623..4432
                     /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(3690..3709)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4579..5167
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5068..5087
                     /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 aaagtggtta attaaggatc ggcaataaaa agacagaata
      781 aaacgcacgg gtgttgggtc gtttgttctg taaaacgacg gccagtgaat tctaccgggt
      841 aggggaggcg cttttcccaa ggcagtctgg agcatgcgct ttagcagccc cgctgggcac
      901 ttggcgctac acaagtggcc tctggcctcg cacacattcc acatccaccg gtaggcgcca
      961 accggctccg ttctttggtg gccccttcgc gccaccttct actcctcccc tagtcaggaa
     1021 gttccccccc gccccgcagc tcgcgtcgtg caggacgtga caaatggaag tagcacgtct
     1081 cactagtctc gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag gcctttgggg
     1141 cagcggccaa tagcagcttt gctccttcgc tttctgggct cagaggctgg gaaggggtgg
     1201 gtccgggggc gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg
     1261 aggcccggca ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct cctcttcctc
     1321 atctccgggc ctttcgaccg atcatcaagc ttgatcctca tgaccgagta caagcccacg
     1381 gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc
     1441 gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc
     1501 gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg
     1561 gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg
     1621 ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa
     1681 cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc
     1741 gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga
     1801 gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac
     1861 ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga
     1921 ccgcgcacct ggtgcatgac ccgcaagccc ggtgccggat ccatgcccac gctactgcgg
     1981 gtttatatag acggtcccca cgggatgggg aaaaccacca ccacgcaact gctggtggcc
     2041 ctgggttcgc gcgacgatat cgtctacgta cccgagccga tgacttactg gcgggtgctg
     2101 ggggcttccg agacaatcgc gaacatctac accacacaac accgcctcga ccagggtgag
     2161 atatcggccg gggacgcggc ggtggtaatg acaagcgccc agataacaat gggcatgcct
     2221 tatgccgtga ccgacgccgt tctggctcct catatcgggg gggaggctgg gagctcacat
     2281 gccccgcccc cggccctcac cctcatcttc gaccgccatc ccatcgccgc cctcctgtgc
     2341 tacccggccg cgcggtacct tatgggcagc atgacccccc aggccgtgct ggcgttcgtg
     2401 gccctcatcc cgccgacctt gcccggcacc aacatcgtgc ttggggccct tccggaggac
     2461 agacacatcg accgcctggc caaacgccag cgccccggcg agcggctgga cctggctatg
     2521 ctggctgcga ttcgccgcgt ttacgggcta cttgccaata cggtgcggta tctgcagtgc
     2581 ggcgggtcgt ggcgggagga ctggggacag ctttcgggga cggccgtgcc gccccagggt
     2641 gccgagcccc agagcaacgc gggcccacga ccccatatcg gggacacgtt atttaccctg
     2701 tttcgggccc ccgagttgct ggcccccaac ggcgacctgt ataacgtgtt tgcctgggcc
     2761 ttggacgtct tggccaaacg cctccgttcc atgcacgtct ttatcctgga ttacgaccaa
     2821 tcgcccgccg gctgccggga cgccctgctg caacttacct ccgggatggt ccagacccac
     2881 gtcaccaccc ccggctccat accgacgata tgcgacctgg cgcgcacgtt tgcccgggag
     2941 atgggggagg ctaactgagc tctagcttat cgactcgccg atagtggaaa ccgacgcccc
     3001 agcactcgtc cgagggcaaa ggaatagtcg agaaattgat gatctattaa acaataaaga
     3061 tgtccacatg gaagtttttc ctgtcatact ttgttaagaa gggtgagaac agagtaccta
     3121 cattttgaat ggaaggattg gagctacggg ggtgggggtg gggtgggatt agataaatgc
     3181 ctgctcttta ctgaaggctc tttactattg ctttatgata atgtttcata gttggatatc
     3241 ataatttaaa caagcaaaac caaattaagg gccagctcat tcctcccact catgatctat
     3301 agatcctcta gagtcgacct gcaggcatgc acccgggcaa ctttattata caaagttggc
     3361 attataaaaa agcattgctt atcaatttgt tgcaacgaac aggtcactat cagtcaaaat
     3421 aaaatcatta tttggagctc catggtagcg ttaacgcggc cgcgatatcc cctatagtga
     3481 gtcgtattac atggtcatag ctgtttcctg gcagctctgg cccgtgtctc aaaatctctg
     3541 atgttacatt gcacaagata aaaatatatc atcatgaaca ataaaactgt ctgcttacat
     3601 aaacagtaat acaaggggtg ttatgagcca tattcaacgg gaaacgtcga ggccgcgatt
     3661 aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca
     3721 atcaggtgcg acaatctatc gcttgtatgg gaagcccgat gcgccagagt tgtttctgaa
     3781 acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct
     3841 gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg
     3901 gttactcacc actgcgatcc ccggaaaaac agcattccag gtattagaag aatatcctga
     3961 ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc
     4021 tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg
     4081 aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt
     4141 tgaacaagtc tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac
     4201 tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat
     4261 tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg
     4321 cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa
     4381 tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct aatcagaatt
     4441 ggttaattgg ttgtaacact ggcagagcat tacgctgact tgacgggacg gcgcaagctc
     4501 atgaccaaaa tcccttaacg tgagttacgc gtcgttccac tgagcgtcag accccgtaga
     4561 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     4621 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     4681 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     4741 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     4801 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     4861 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     4921 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     4981 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     5041 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     5101 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     5161 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     5221 tcacatgtt
//
