LOCUS       sequence-64402-e        4764 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 4764)
  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 4764)
  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-hygro-amp
FEATURES             Location/Qualifiers
     source          1..4764
                     /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"
     CDS             complement(join(2090..2881,2882..2950))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2713..2732
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     protein_bind    3012..3028
                     /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     3017..3039
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3036..3052
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3036..3052
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3101..3222
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3112..3131)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3166..3185
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3352..4377)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(3352..4377)
                     /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(4426..4622)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        complement(4434..4644)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4440..4575
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4494..4513)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     protein_bind    complement(4722..4755)
                     /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 gaacagtatt tggtatctgc gctctgctga
     1801 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaaaaa accaccgctg
     1861 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     1921 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     1981 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     2041 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     2101 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     2161 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     2221 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     2281 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     2341 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     2401 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     2461 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     2521 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     2581 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     2641 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     2701 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     2761 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     2821 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     2881 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     2941 gaatactcat actcttcctt tttcaatatt caggcttacc tttatgcttc cggctcgtat
     3001 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
     3061 cgccaagctc tagctagagg tcgacggtat acagacatga taagatacat tgatgagttt
     3121 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
     3181 attgctttat ttgtaaccat tataagctgc aataaacaag ttggggtggg cgaagaactc
     3241 cagcatgaga tccccgcgct ggaggatcat ccagccggcg tcccggaaaa cgattccgaa
     3301 gcccaacctt tcatagaagg cggcggtgga atcgaaatct cgtagcacgt gctattcctt
     3361 tgccctcgga cgagtgctgg ggcgtcggtt tccactatcg gcgagtactt ctacacagcc
     3421 atcggtccag acggccgcgc ttctgcgggc gatttgtgta cgcccgacag tcccggctcc
     3481 ggatcggacg attgcgtcgc atcgaccctg cgcccaagct gcatcatcga aattgccgtc
     3541 aaccaagctc tgatagagtt ggtcaagacc aatgcggagc atatacgccc ggagccgcgg
     3601 cgatcctgca agctccggat gcctccgctc gaagtagcgc gtctgctgct ccatacaagc
     3661 caaccacggc ctccagaaga agatgttggc gacctcgtat tgggaatccc cgaacatcgc
     3721 ctcgctccag tcaatgaccg ctgttatgcg gccattgtcc gtcaggacat tgttggagcc
     3781 gaaatccgcg tgcacgaggt gccggacttc ggggcagtcc tcggcccaaa gcatcagctc
     3841 atcgagagcc tgcgcgacgg acgcactgac ggtgtcgtcc atcacagttt gccagtgata
     3901 cacatgggga tcagcaatcg cgcatatgaa atcacgccat gtagtgtatt gaccgattcc
     3961 ttgcggtccg aatgggccga acccgctcgt ctggctaaga tcggccgcag cgatcgcatc
     4021 catggcctcc gcgaccggct gcagaacagc gggcagttcg gtttcaggca ggtcttgcaa
     4081 cgtgacaccc tgtgcacggc gggagatgca ataggtcagg ctctcgctga attccccaat
     4141 gtcaagcact tccggaatcg ggagcgcggc cgatgcaaag tgccgataaa cataacgatc
     4201 tttgtagaaa ccatcggcgc agctatttac ccgcaggaca tatccacgcc ctcctacatc
     4261 gaagctgaaa gcacgagatt cttcgccctc cgagagctgc atcaggtcgg agacgctgtc
     4321 gaacttttcg atcagaaact tctcgacaga cgtcgcggtg agttcaggct ttttcatcac
     4381 gtgctgatca gatccgaaaa tggatataca agctcccggg agctttttgc aaaagcctag
     4441 gcctccaaaa aagcctcccc actacttctg gaatagctca gaggcagagg cggcctcggc
     4501 ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg aactgggcgg
     4561 agttaggggc gggatgggcg gagttagggg cgggactatg gttgctgact aattgagatg
     4621 catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc tggttgctaa
     4681 ctaattgaga tgcatgcttt gcatacttct gcctgctgga aataacttcg tatagcatac
     4741 attatacgaa cggtactcga gggg
//
