LOCUS       sequence-64408-e        4710 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4710)
  AUTHORS   Orlowska KP, Klosowska K, Szczesny RJ, Cysewski D, Krawczyk PS, 
            Dziembowski A
  TITLE     A new strategy for gene targeting and functional proteomics using 
            the DT40 cell line.
  JOURNAL   Nucleic Acids Res. 2013 Sep;41(17):e167. doi: 10.1093/nar/gkt650. 
            Epub 2013 Jul 27.
  PUBMED    23892402
REFERENCE   2  (bases 1 to 4710)
  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: "Nucleic 
            Acids Res."; date: "2013-09"; volume: "41(17):e167. doi"; pages: " 
            10.1093/nar/gkt650. Epub 2013 Jul 27"
COMMENT     Sequence Label: pQuantEGFP-puro-kana
FEATURES             Location/Qualifiers
     source          1..4710
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             37..57
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(64..66,67..69,70..783)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(64..66,67..69,70..780)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(109..130)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(370..389)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     717..738
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(798..831)
                     /label=lox71
                     /bound_moiety="Cre recombinase"
                     /note="Left element (LE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(1466..1483)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1637..2225)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1717..1736)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2336..2355)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2347..3162
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             2347..3162
                     /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="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2420..2439)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     protein_bind    3239..3255
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     3244..3266
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3263..3279
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3263..3279
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3365..3446
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     3390..3409
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3676..4275)
                     /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(3759..3779)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     4256..4275
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(4372..4568)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        complement(4380..4590)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4386..4521
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4440..4459)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     protein_bind    complement(4668..4701)
                     /label=lox66
                     /bound_moiety="Cre recombinase"
                     /note="Right element (RE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 aagcttgccg ccgatatcac cagcctgtac aagggcgaga atttgtattt tcagggtgag
       61 ctcatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
      121 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
      181 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
      241 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
      301 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
      361 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
      421 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
      481 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
      541 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
      601 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
      661 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
      721 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
      781 taaactagtg gatcccctac cgttcgtata gcatacatta tacgaagtta ttgatcaacc
      841 ggactgttac caacacccac acccctgtga tgaaacaaaa cccataaatg cgcataaaac
      901 aagacgagat tggcatggct ttatttgttt tttcttttgg cgcttgactc aggattaaaa
      961 aactggaatg gtgaaggtgt cagcagcagt cttaaaatga aacatgttgg agcgaacgcc
     1021 cccaaagttc tacaatgcat ctgaggactt tgattgtaca tttgtttctt ttttaatagt
     1081 cattccaaat attgttataa tgcattgtta caggaagtta ctcgcctctg tgaaggcaac
     1141 agcccagctg ggaggagccg gtaccaatta ctggtgttag atgataattg cttgtctgta
     1201 aattatgtaa cccaacaagt gtctttttgt atcttccgcc ttaaaaacaa aacacacttg
     1261 atcctttttg gtttgtcaag caagcgggct gtgttcccca gtgatagatg tgaatgaagg
     1321 ctttacagtc ccccacagtc taggagtaaa gtgccagtat gtgggggagg gaggggctac
     1381 ctgtacactg acttaagacc agttcaaata aaagtgcaca caatagaggc ttgactggtg
     1441 ttcccagggg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     1501 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     1561 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     1621 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     1681 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     1741 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     1801 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     1861 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     1921 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     1981 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     2041 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     2101 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     2161 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     2221 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     2281 gttaagggat tttggtcatg aacaataaaa ctgtctgctt acataaacag taatacaagg
     2341 ggtgttatga gccatattca acgggaaacg tcttgctcta ggccgcgatt aaattccaac
     2401 atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg
     2461 acaatctatc gattgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa
     2521 ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt
     2581 atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc
     2641 actgcgatcc ccgggaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa
     2701 aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat
     2761 tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac
     2821 ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc
     2881 tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat
     2941 ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga
     3001 cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag
     3061 ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg
     3121 aataaattgc agtttcattt gatgctcgat gagtttttct aagaattaat tcatgaggct
     3181 cttccttttt caatattcag gcttaccttt atgcttccgg ctcgtatgtt gtgtggaatt
     3241 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagctctag
     3301 ctagaggtcg acaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     3361 gattgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     3421 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg
     3481 ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg
     3541 ctgattatga tcctctagag tcggtgggcc tcgggggcgg gtgcggggtc ggcggggccg
     3601 ccccgggtgg cttcggtcgg agccatgggg tcgtgcgctc ctttcggtcg ggcgctgcgg
     3661 gtcgtggggc gggcgtcagg caccgggctt gcgggtcatg caccaggtgc gcggtccttc
     3721 gggcacctcg acgtcggcgg tgacggtgaa gccgagccgc tcgtagaagg ggaggttgcg
     3781 gggcgcggag gtctccagga aggcgggcac cccggcgcgc tcggccgcct ccactccggg
     3841 gagcacgacg gcgctgccca gacccttgcc ctggtggtcg ggcgagacgc cgacggtggc
     3901 caggaaccac gcgggctcct tgggccggtg cggcgccagg aggccttcca tctgttgctg
     3961 cgcggccagc cgggaaccgc tcaactcggc catgcgcggg ccgatctcgg cgaacaccgc
     4021 ccccgcttcg acgctctccg gcgtggtcca gaccgccacc gcggcgccgt cgtccgcgac
     4081 ccacaccttg ccgatgtcga gcccgacgcg cgtgaggaag agttcttgca gctcggtgac
     4141 ccgctcgatg tggcggtccg ggtcgacggt gtggcgcgtg gcggggtagt cggcgaacgc
     4201 ggcggcgagg gtgcgtacgg cccgggggac gtcgtcgcgg gtggcgaggc gcaccgtggg
     4261 cttgtactcg gtcatggaag gtcgtctcct tgtgaggggt caggggcgtg ggtcagggga
     4321 tggtggcggc accggtcgtg gcggccgacc tgcaggcatg caagctagct ttttgcaaaa
     4381 gcctaggcct ccaaaaaagc ctccccacta cttctggaat agctcagagg cagaggcggc
     4441 ctcggcctct gcataaataa aaaaaattag tcagccatgg ggcggagaat gggcggaact
     4501 gggcggagtt aggggcggga tgggcggagt taggggcggg actatggttg ctgactaatt
     4561 gagatgcatg ctttgcatac ttctgcctgc tggggagcct ggggactttc cacacctggt
     4621 tgctaactaa ttgagatgca tgctttgcat acttctgcct gctggaaata acttcgtata
     4681 gcatacatta tacgaacggt actcgagggg
//
