LOCUS       sequence-64404-e        4404 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 4404)
  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 4404)
  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: pQuantA-puro-kana
FEATURES             Location/Qualifiers
     source          1..4404
                     /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             97..270
                     /codon_start=1
                     /product="IgG-binding unit of Staphylococcus aureus protein
                     A"
                     /label=ProtA
                     /translation="VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLL
                     AEAKKLNDAQAPK"
     CDS             271..444
                     /codon_start=1
                     /product="IgG-binding unit of Staphylococcus aureus protein
                     A"
                     /label=ProtA
                     /translation="VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLL
                     AEAKKLNGAQAPK"
     protein_bind    complement(492..525)
                     /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(1160..1177)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1331..1919)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1411..1430)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2030..2049)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2041..2856
                     /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             2041..2856
                     /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(2114..2133)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     protein_bind    2933..2949
                     /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     2938..2960
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2957..2973
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2957..2973
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3059..3140
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     3084..3103
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3370..3969)
                     /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(3453..3473)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     3950..3969
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(4066..4262)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        complement(4074..4284)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4080..4215
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4134..4153)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     protein_bind    complement(4362..4395)
                     /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 ctcaaaaccg cggctcttgc gcaacacgat gaagccgtgg acaacaaatt caacaaagaa
      121 caacaaaacg cgttctatga gatcttacat ttacctaact taaacgaaga acaacgaaac
      181 gccttcatcc aaagtttaaa agatgaccca agccaaagcg ctaacctttt agcggaagct
      241 aaaaagctaa atgatgctca ggcgccgaaa gtagacaaca aattcaacaa agaacaacaa
      301 aacgcgttct atgagatctt acatttacct aacttaaacg aagaacaacg aaacgccttc
      361 atccaaagtt taaaagatga cccaagccaa agcgctaacc ttttagcaga agctaaaaag
      421 ctaaatggtg ctcaggcgcc gaaagtagac gcgaattccg cggggaagtc aacctgaact
      481 agtggatccc ctaccgttcg tatagcatac attatacgaa gttattgatc aaccggactg
      541 ttaccaacac ccacacccct gtgatgaaac aaaacccata aatgcgcata aaacaagacg
      601 agattggcat ggctttattt gttttttctt ttggcgcttg actcaggatt aaaaaactgg
      661 aatggtgaag gtgtcagcag cagtcttaaa atgaaacatg ttggagcgaa cgcccccaaa
      721 gttctacaat gcatctgagg actttgattg tacatttgtt tcttttttaa tagtcattcc
      781 aaatattgtt ataatgcatt gttacaggaa gttactcgcc tctgtgaagg caacagccca
      841 gctgggagga gccggtacca attactggtg ttagatgata attgcttgtc tgtaaattat
      901 gtaacccaac aagtgtcttt ttgtatcttc cgccttaaaa acaaaacaca cttgatcctt
      961 tttggtttgt caagcaagcg ggctgtgttc cccagtgata gatgtgaatg aaggctttac
     1021 agtcccccac agtctaggag taaagtgcca gtatgtgggg gagggagggg ctacctgtac
     1081 actgacttaa gaccagttca aataaaagtg cacacaatag aggcttgact ggtgttccca
     1141 gggggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     1201 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     1261 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     1321 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     1381 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     1441 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     1501 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     1561 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     1621 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     1681 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     1741 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga
     1801 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     1861 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     1921 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     1981 ggattttggt catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt
     2041 atgagccata ttcaacggga aacgtcttgc tctaggccgc gattaaattc caacatggat
     2101 gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc
     2161 tatcgattgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc
     2221 gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct
     2281 cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg
     2341 atccccggga aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt
     2401 gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct
     2461 tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg
     2521 gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa
     2581 gaaatgcata aacttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca
     2641 cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc
     2701 ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct
     2761 ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa
     2821 ttgcagtttc atttgatgct cgatgagttt ttctaagaat taattcatga ggctcttcct
     2881 ttttcaatat tcaggcttac ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
     2941 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct ctagctagag
     3001 gtcgacaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgagattgc
     3061 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     3121 taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg
     3181 gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt atggctgatt
     3241 atgatcctct agagtcggtg ggcctcgggg gcgggtgcgg ggtcggcggg gccgccccgg
     3301 gtggcttcgg tcggagccat ggggtcgtgc gctcctttcg gtcgggcgct gcgggtcgtg
     3361 gggcgggcgt caggcaccgg gcttgcgggt catgcaccag gtgcgcggtc cttcgggcac
     3421 ctcgacgtcg gcggtgacgg tgaagccgag ccgctcgtag aaggggaggt tgcggggcgc
     3481 ggaggtctcc aggaaggcgg gcaccccggc gcgctcggcc gcctccactc cggggagcac
     3541 gacggcgctg cccagaccct tgccctggtg gtcgggcgag acgccgacgg tggccaggaa
     3601 ccacgcgggc tccttgggcc ggtgcggcgc caggaggcct tccatctgtt gctgcgcggc
     3661 cagccgggaa ccgctcaact cggccatgcg cgggccgatc tcggcgaaca ccgcccccgc
     3721 ttcgacgctc tccggcgtgg tccagaccgc caccgcggcg ccgtcgtccg cgacccacac
     3781 cttgccgatg tcgagcccga cgcgcgtgag gaagagttct tgcagctcgg tgacccgctc
     3841 gatgtggcgg tccgggtcga cggtgtggcg cgtggcgggg tagtcggcga acgcggcggc
     3901 gagggtgcgt acggcccggg ggacgtcgtc gcgggtggcg aggcgcaccg tgggcttgta
     3961 ctcggtcatg gaaggtcgtc tccttgtgag gggtcagggg cgtgggtcag gggatggtgg
     4021 cggcaccggt cgtggcggcc gacctgcagg catgcaagct agctttttgc aaaagcctag
     4081 gcctccaaaa aagcctcccc actacttctg gaatagctca gaggcagagg cggcctcggc
     4141 ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg aactgggcgg
     4201 agttaggggc gggatgggcg gagttagggg cgggactatg gttgctgact aattgagatg
     4261 catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc tggttgctaa
     4321 ctaattgaga tgcatgcttt gcatacttct gcctgctgga aataacttcg tatagcatac
     4381 attatacgaa cggtactcga gggg
//
