LOCUS       sequence-211550-        4633 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Puromycin resistant cassette for knock-in by VIKING method.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4633)
  AUTHORS   Sawatsubashi S, Joko Y, Fukumoto S, Matsumoto T, Sugano SS
  TITLE     Development of versatile non-homologous end joining-based knock-in 
            module for genome editing.
  JOURNAL   Sci Rep. 2018 Jan 12;8(1):593. doi: 10.1038/s41598-017-18911-9.
  PUBMED    29330493
REFERENCE   2  (bases 1 to 4633)
  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"; journalName: "Sci Rep."; 
            date: "2018-01-12"; volume: "8(1):593. doi"; pages: " 
            10.1038/s41598-017-18911-9"
COMMENT     Sequence Label: pVKG1-Puro
FEATURES             Location/Qualifiers
     source          1..4633
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..212
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             225..493
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             520..1119
                     /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(520..539)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1016..1036
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    1126..1714
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1179..1199)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1597..1608)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             1788..2021
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2093..2214
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2130..2149)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2184..2203
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2231..2250)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2232..2250)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2255..2271)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2255..2271)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2373..2392)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2384..3193
                     /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(2451..2470)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3340..3928
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4082..4099
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4258..4285)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4377..4463)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     4512..4534
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4526..4543
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4527..4543
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa
       61 cgggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
      121 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
      181 tttttcgcaa cgggtttgcc gccagaacac agctgaagct tcgaggggct cgcatctctc
      241 cttcacgcgc ccgccgccct acctgaggcc gccatccacg ccggttgagt cgcgttctgc
      301 cgcctcccgc ctgtggtgcc tcctgaactg cgtccgccgt ctaggtaagt ttaaagctca
      361 ggtcgagacc gggcctttgt ccggcgctcc cttggagcct acctagactc agccggctct
      421 ccacgctttg cctgaccctg cttgctcaac tctacgtctt tgtttcgttt tctgttctgc
      481 gccgttacag atccaagctg tgaccggcgc ctacgctaga tgaccgagta caagcccacg
      541 gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc
      601 gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc
      661 gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg
      721 gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg
      781 ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa
      841 cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc
      901 gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga
      961 gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac
     1021 ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga
     1081 ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgag tcgacaatca acctctggat
     1141 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     1201 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     1261 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
     1321 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
     1381 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
     1441 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     1501 tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc
     1561 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
     1621 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag
     1681 acgagtcgga tctccctttg ggccgcctcc ccgcctggta cctttaagac caatgactta
     1741 caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat
     1801 tcactcccaa cgaagataag atctgctttt tgcttgtact gggtctctct ggttagacca
     1861 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag
     1921 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag
     1981 atccctcaga cccttttagt cagtgtggaa aatctctagc agtagtagtt catgtcatct
     2041 tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt
     2101 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     2161 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatttaat
     2221 taagtttatc ccctatagtg agtcgtatta catggtcata gctgtttcct ggcagctctg
     2281 gcccgtgtct caaaatctct gatgttacat tgcacaagat aaaaatatat catcatgaac
     2341 aataaaactg tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg
     2401 ggaaacgtcg aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg
     2461 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgcttgtatg ggaagcccga
     2521 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga
     2581 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat
     2641 ccgtactcct gatgatgcat ggttactcac cactgcgatc cccggaaaaa cagcattcca
     2701 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct
     2761 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg
     2821 tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga
     2881 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt
     2941 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga
     3001 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga
     3061 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt
     3121 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga
     3181 tgagtttttc taatcagaat tggttaattg gttgtaacac tggcagagca ttacgctgac
     3241 ttgacgggac ggcgcaagct catgaccaaa atcccttaac gtgagttacg cgtcgttcca
     3301 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     3361 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     3421 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     3481 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     3541 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     3601 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     3661 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     3721 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     3781 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     3841 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     3901 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     3961 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     4021 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     4081 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     4141 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     4201 tgagcgcaac gcaattaata cgcgtaccgc tagacaggaa gagtttgtag aaacgcaaaa
     4261 aggccatccg tcaggatggc cttctgctta gtttgatgcc tggcagttta tggcgggcgt
     4321 cctgcccgcc accctccggg ccgttgcttc acaacgttca aatccgctcc cggcggattt
     4381 gtcctactca ggagagcgtt caccgacaaa caacagataa aacgaaaggc ccagtcttcc
     4441 gactgagcct ttcgttttat ttgatgcctg gcagttccct actctcgcgt taacgctagg
     4501 atggatgttt tcccagtcac gacgttgtaa aacgacggcc agtcttaagc tcgggccccg
     4561 ctagcaagct ttttgaattc cgcggtggcg cgccggccgc gaaggatctg cgatcgctcc
     4621 ggtgcccgtc agt
//
