LOCUS       sequence-64412-e        4298 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 4298)
  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 4298)
  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-hygro-kana
FEATURES             Location/Qualifiers
     source          1..4298
                     /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    2635..2756
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2646..2665)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2700..2719
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(2886..3911)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(2886..3911)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     promoter        complement(3960..4156)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        complement(3968..4178)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3974..4109
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4028..4047)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     protein_bind    complement(4256..4289)
                     /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 gaggtcgacg gtatacagac atgataagat
     2641 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     2701 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttgggg
     2761 tgggcgaaga actccagcat gagatccccg cgctggagga tcatccagcc ggcgtcccgg
     2821 aaaacgattc cgaagcccaa cctttcatag aaggcggcgg tggaatcgaa atctcgtagc
     2881 acgtgctatt cctttgccct cggacgagtg ctggggcgtc ggtttccact atcggcgagt
     2941 acttctacac agccatcggt ccagacggcc gcgcttctgc gggcgatttg tgtacgcccg
     3001 acagtcccgg ctccggatcg gacgattgcg tcgcatcgac cctgcgccca agctgcatca
     3061 tcgaaattgc cgtcaaccaa gctctgatag agttggtcaa gaccaatgcg gagcatatac
     3121 gcccggagcc gcggcgatcc tgcaagctcc ggatgcctcc gctcgaagta gcgcgtctgc
     3181 tgctccatac aagccaacca cggcctccag aagaagatgt tggcgacctc gtattgggaa
     3241 tccccgaaca tcgcctcgct ccagtcaatg accgctgtta tgcggccatt gtccgtcagg
     3301 acattgttgg agccgaaatc cgcgtgcacg aggtgccgga cttcggggca gtcctcggcc
     3361 caaagcatca gctcatcgag agcctgcgcg acggacgcac tgacggtgtc gtccatcaca
     3421 gtttgccagt gatacacatg gggatcagca atcgcgcata tgaaatcacg ccatgtagtg
     3481 tattgaccga ttccttgcgg tccgaatggg ccgaacccgc tcgtctggct aagatcggcc
     3541 gcagcgatcg catccatggc ctccgcgacc ggctgcagaa cagcgggcag ttcggtttca
     3601 ggcaggtctt gcaacgtgac accctgtgca cggcgggaga tgcaataggt caggctctcg
     3661 ctgaattccc caatgtcaag cacttccgga atcgggagcg cggccgatgc aaagtgccga
     3721 taaacataac gatctttgta gaaaccatcg gcgcagctat ttacccgcag gacatatcca
     3781 cgccctccta catcgaagct gaaagcacga gattcttcgc cctccgagag ctgcatcagg
     3841 tcggagacgc tgtcgaactt ttcgatcaga aacttctcga cagacgtcgc ggtgagttca
     3901 ggctttttca tcacgtgctg atcagatccg aaaatggata tacaagctcc cgggagcttt
     3961 ttgcaaaagc ctaggcctcc aaaaaagcct ccccactact tctggaatag ctcagaggca
     4021 gaggcggcct cggcctctgc ataaataaaa aaaattagtc agccatgggg cggagaatgg
     4081 gcggaactgg gcggagttag gggcgggatg ggcggagtta ggggcgggac tatggttgct
     4141 gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca
     4201 cacctggttg ctaactaatt gagatgcatg ctttgcatac ttctgcctgc tggaaataac
     4261 ttcgtatagc atacattata cgaacggtac tcgagggg
//
