LOCUS       sequence-64429-e        4474 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 4474)
  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 4474)
  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: pQuantFLAG-bsr-kana
FEATURES             Location/Qualifiers
     source          1..4474
                     /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             64..87
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    complement(105..138)
                     /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(773..790)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(944..1532)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1024..1043)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1643..1662)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1654..2469
                     /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             1654..2469
                     /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(1727..1746)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     protein_bind    2546..2562
                     /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     2551..2573
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2570..2586
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2570..2586
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    2629..2750
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2640..2659)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2694..2713
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3175..3195)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          3325..3390
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     promoter        complement(4136..4332)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        complement(4144..4354)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4150..4285
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4204..4223)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     protein_bind    complement(4432..4465)
                     /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 ctcgattaca aggatgacga cgataagtag actagtggat cccctaccgt tcgtatagca
      121 tacattatac gaagttattg atcaaccgga ctgttaccaa cacccacacc cctgtgatga
      181 aacaaaaccc ataaatgcgc ataaaacaag acgagattgg catggcttta tttgtttttt
      241 cttttggcgc ttgactcagg attaaaaaac tggaatggtg aaggtgtcag cagcagtctt
      301 aaaatgaaac atgttggagc gaacgccccc aaagttctac aatgcatctg aggactttga
      361 ttgtacattt gtttcttttt taatagtcat tccaaatatt gttataatgc attgttacag
      421 gaagttactc gcctctgtga aggcaacagc ccagctggga ggagccggta ccaattactg
      481 gtgttagatg ataattgctt gtctgtaaat tatgtaaccc aacaagtgtc tttttgtatc
      541 ttccgcctta aaaacaaaac acacttgatc ctttttggtt tgtcaagcaa gcgggctgtg
      601 ttccccagtg atagatgtga atgaaggctt tacagtcccc cacagtctag gagtaaagtg
      661 ccagtatgtg ggggagggag gggctacctg tacactgact taagaccagt tcaaataaaa
      721 gtgcacacaa tagaggcttg actggtgttc ccagggggcg ctcttccgct tcctcgctca
      781 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
      841 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
      901 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
      961 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     1021 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     1081 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     1141 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     1201 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     1261 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     1321 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     1381 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     1441 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     1501 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     1561 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaac aataaaactg
     1621 tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtct
     1681 tgctctaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct
     1741 cgcgataatg tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg
     1801 ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg
     1861 gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt
     1921 actcctgatg atgcatggtt actcaccact gcgatccccg ggaaaacagc attccaggta
     1981 ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc
     2041 cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc
     2101 gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag
     2161 cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca
     2221 ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg
     2281 aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt
     2341 gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa
     2401 aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag
     2461 tttttctaag aattaattca tgaggctctt cctttttcaa tattcaggct tacctttatg
     2521 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     2581 tatgaccatg attacgccaa gctctagcta gaggtcgacc agacatgata agatacattg
     2641 atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt
     2701 gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca
     2761 attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt
     2821 aaaacctcta caaatgtggt atggctgatt atgatctcta gtcaaggcac tatacatcaa
     2881 atattcctta ttaacccctt tacaaattaa aaagctaaag gtacacaatt tttgagcata
     2941 gttattaata gcagacactc tatgcctgtg tggagtaaga aaaaacagta tgttatgatt
     3001 ataactgtta tgcctactta taaaggttac agaatatttt tccataattt tcttgtatag
     3061 cagtgcagct ttttcctttg tggtgtaaat agcaaagcaa gcaagagttc tattactaaa
     3121 cacagcatga ctcaaaaaac ttagcaattc tgaaggaaag tccttggggt cttctacctt
     3181 tctcttcttt tttggaggag tagaatgttg agagtcagca gtagcctcat catcactaga
     3241 tggcatttct tctgagcaaa acaggttttc ctcattaaag gcattccacc actgctccca
     3301 ttcatcagtt ccataggttg gaatctaaaa tacacaaaca attagaatca gtagtttaac
     3361 acattataca cttaaaaatt ttatatttac cttagagctt taaatctctg taggtagttt
     3421 gtccaattat gtcacaccac agaagtaagg ttccttcaca aagatccgga ccaaagcggc
     3481 catcgtgcct ccccactcct gcagttcggg ggcatggatg cgcggatagc cgctgctggt
     3541 ttcctggatg ccgacggatt tgcactgccg gtagaactcc gcgaggtcgt ccagcctcag
     3601 gcagcagcca agctagcttg gtatggtaaa acttttaatt tcgggtatat ttgagtggaa
     3661 tgagttcttc aatcgtagtt ttgactaact tgccattcat ttctattaac acaaaacaat
     3721 ctggtgcata gtctgaaatc aactccctac acataccaca aggacttacc actcgaatac
     3781 ttctatctac ttcgtcagaa taagggtgtc taacagctac aatcgtgtca aaatcctttt
     3841 gtccattcga aactgcacta ccaatcgcaa tggcttctgc acaaacagtt actcgtccta
     3901 tatacgcttc aatatgtact gccgaaatga tttctcctgt tttcgtacga attgccgctc
     3961 ccacatgatg tttattatcc tcataaagca ttgtaatctt ctctgtcgct acttctacta
     4021 attctagatc ttgttgagaa atgttaaatg ttttcatata ttcttcttcc tccgatttct
     4081 gcaaaaatga atccaccggt cgtggcggcc gacctgcagg catgcaagct agctttttgc
     4141 aaaagcctag gcctccaaaa aagcctcccc actacttctg gaatagctca gaggcagagg
     4201 cggcctcggc ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg
     4261 aactgggcgg agttaggggc gggatgggcg gagttagggg cgggactatg gttgctgact
     4321 aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc
     4381 tggttgctaa ctaattgaga tgcatgcttt gcatacttct gcctgctgga aataacttcg
     4441 tatagcatac attatacgaa cggtactcga gggg
//
