LOCUS       sequence-12659-e        6318 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 6318)
  AUTHORS   Osterwalder M, Galli A, Rosen B, Skarnes WC, Zeller R, Lopez-Rios J.
  TITLE     Dual RMCE for efficient re-engineering of mouse mutant alleles
  JOURNAL   Nat Methods. 2010 Oct 17
  PUBMED    20953177
REFERENCE   2  (bases 1 to 6318)
  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: "Nat 
            Methods. 2010 Oct 17"
COMMENT     Sequence Label: pDREV-0
FEATURES             Location/Qualifiers
     source          1..6318
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      103..189
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      281..308
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     316..339
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     protein_bind    358..457
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    470..517
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     misc_feature    join(1309..1392,1393..1424)
                     /label=En2 SA
                     /note="splice acceptor from the mouse Engrailed 2 gene 
                     (Skarnes et al., 1992)"
     CDS             1435..1488
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1495..1869
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="PEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSIYVYKVL
                     KQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPG
                     ELAKHAVSEGTKAVTKYTSAK"
     CDS             join(1876..1878,1879..1881,1882..2595)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1921..1942)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2182..2201)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2529..2550
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2718..2852
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2755..2774)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2809..2828
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    2862..2893
                     /label=rox
                     /bound_moiety="Dre recombinase"
     promoter        2935..3434
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2970..2993)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3367..3386
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3474..4073
                     /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(3474..3493)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3970..3990
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    4614..4645
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    complement(4654..4687)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(4709..4808)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        4907..4954
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4973..5545
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     rep_origin      5668..6256
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6157..6176
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgctagca tggatctcgg
       61 ggacgtctaa ctactaagcg agagtaggga actgccaggc atcaaataaa acgaaaggct
      121 cagtcggaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
      181 aggacaaatc cgccgggagc ggatttgaac gttgtgaagc aacggcccgg agggtggcgg
      241 gcaggacgcc cgccataaac tgccaggcat caaactaagc agaaggccat cctgacggat
      301 ggcctttttg cgtttctaca aactcttcct gttagttagt tacttaagct cgggccccaa
      361 ataatgattt tattttgact gatagtgacc tgttcgttgc aacaaattga taagcaatgc
      421 ttttttataa tgccaacttt gtacaaaaaa gcaggctggc gccggaaccg aagttcctat
      481 tccgaagttc ctattctcta gaaagtatag gaacttcgaa ccctttccca caccaccctc
      541 cacacttgcc ccaaacactg ccaactatgt aggaggaagg ggttgggact aacagaagaa
      601 cccgttgtgg ggaagctgtt gggagggtca ctttatgttc ttgcccaagg tcagttgggt
      661 ggcctgcttc tgatgaggtg gtcccaaggt ctggggtaga aggtgagagg gacaggccac
      721 caaggtcagc cccccccccc tatcccatag gagccaggtc cctctcctgg acaggaagac
      781 tgaaggggag atgccagaga ctcagtgaag cctggggtac cctattggag tccttcaagg
      841 aaacaaactt ggcctcacca ggcctcagcc ttggctcctc ctgggaactc tactgccctt
      901 gggatcccct tgtagttgtg ggttacatag gaagggggac gggattcccc ttgactggct
      961 agcctactct tttcttcagt cttctccatc tcctctcacc tgtctctcga ccctttccct
     1021 aggatagact tggaaaaaga taaggggaga aaacaaatgc aaacgaggcc agaaagattt
     1081 tggctgggca ttccttccgc tagcttttat tgggatcccc tagtttgtga taggcctttt
     1141 agctacatct gccaatccat ctcattttca cacacacaca caccactttc cttctggtca
     1201 gtgggcacat gtccagcctc aagtttatat caccaccccc aatgcccaac acttgtatgg
     1261 ccttgggcgg gtcatccccc cccccacccc cagtatctgc aacctcaagc tagcttgggt
     1321 gcgttggttg tggataagta gctagactcc agcaaccagt aacctctgcc ctttctcctc
     1381 catgacaacc aggtcccagg tcccgaaaac caaagaagaa gaacgcagat ctccgagggc
     1441 agaggaagtc ttctaacatg cggtgacgtg gaggagaatc ccggaccggg gggaccagag
     1501 ccagcgaagt ctgctcccgc cccgaaaaag ggctccaaga aggcggtgac taaggcgcag
     1561 aagaaagacg gcaagaagcg caagcgcagc cgcaaggaga gctattccat ctatgtgtac
     1621 aaggttctga agcaggtcca ccctgacacc ggcatttcgt ccaaggccat gggcatcatg
     1681 aattcgtttg tgaacgacat tttcgagcgc atcgcaggtg aggcttcccg cctggcgcat
     1741 tacaacaagc gctcgaccat cacctccagg gagatccaga cggccgtgcg cctgctgctg
     1801 cctggggagt tggccaagca cgccgtgtcc gagggtacta aggccgtcac caagtacacc
     1861 agcgctaaat ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     1921 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     1981 ggcgatgcca cctacggcaa gctgaccctg aagctgatct gcaccaccgg caagctgccc
     2041 gtgccctggc ccaccctcgt gaccaccctg ggctacggcc tgcagtgctt cgcccgctac
     2101 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     2161 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     2221 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     2281 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcaccgcc
     2341 gacaagcaga agaacggcat caaggccaac ttcaagatcc gccacaacat cgaggacggc
     2401 ggcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     2461 ctgcccgaca accactacct gagctaccag tccgccctga gcaaagaccc caacgagaag
     2521 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     2581 gagctgtaca agtaagaaga gcttaattaa tgatcataat cagccatatc acatctgtag
     2641 aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga
     2701 atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     2761 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     2821 aactcatcaa tgtatcttat catgtctgga tccgtttaaa ctaactttaa ataattggca
     2881 ttatttaaag ttacatatgg cggccgccct aaggctcgac ctcgaaattc taccgggtag
     2941 gggaggcgct tttcccaagg cagtctggag catgcgcttt agcagccccg ctgggcactt
     3001 ggcgctacac aagtggcctc tggcctcgca cacattccac atccaccggt aggcgccaac
     3061 cggctccgtt ctttggtggc cccttcgcgc caccttctac tcctccccta gtcaggaagt
     3121 tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca aatggaagta gcacgtctca
     3181 ctagtctcgt gcagatggac agcaccgctg agcaatggaa gcgggtaggc ctttggggca
     3241 gcggccaata gcagctttgc tccttcgctt tctgggctca gaggctggga aggggtgggt
     3301 ccgggggcgg gctcaggggc gggctcaggg gcggggcggg cgcccgaagg tcttccggag
     3361 gcccggcatt ctgcacgctt caaaagcgca cgtctgccgc gctgttctcc tcttcctcat
     3421 ctccgggcct ttcgacctgc atccatctag atctcgagca gctgaagctt accatgaccg
     3481 agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc
     3541 tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatcca gatcgccaca
     3601 tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca
     3661 aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg
     3721 aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc
     3781 tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt
     3841 ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg
     3901 tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct
     3961 ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg
     4021 aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgacgcccgc
     4081 cccacgaccc gcagcgcccg accgaaagga gcgcacgacc ccatgcatcg atgatatcag
     4141 atccccggga tgcagaaatt gatgatctat taaacaataa agatgtccac taaaatggaa
     4201 gtttttcctg tcatactttg ttaagaaggg tgagaacaga gtacctacat tttgaatgga
     4261 aggattggag ctacgggggt gggggtgggg tgggattaga taaatgcntg ctctttactg
     4321 aaggctcttt actattgctt tatgataatg tttcatagtt ggatatcata atttaaacaa
     4381 gcaaaaccaa attaagggcc agctcattcc tcccactcat gatctataga tctatagatc
     4441 tctcgtggga tcattgtttt tctcttgatt cccactttgt ggttctaagt actgtggttt
     4501 ccaaatgtgt cagtttcata gcctgaagaa cgagatcagc agcctctgtt ccacatacac
     4561 ttcattctca gtattgtttt gccaagttct aattccatca gaagctggtc gagtaacttt
     4621 aaataattgg cattatttaa agttaggcgc gccataactt cgtatagcat acattatacg
     4681 aagttatgtc gagatattta aatattagac ccagctttct tgtacaaagt tggcattata
     4741 agaaagcatt gcttatcaat ttgttgcaac gaacaggtca ctatcagtca aaataaaatc
     4801 attatttgcc atccagctgc agctctggcc cgtgtctcaa aatctctgat gttacattgc
     4861 acaagataaa agcttgtata tccattttcg gatctgatca gcacgtgttg acaattaatc
     4921 atcggcatag tatatcggca tagtataata cgacaaggtg aggaactaaa ccatgggtac
     4981 cactcttgac gacacggctt accggtaccg caccagtgtc ccgggggacg ccgaggccat
     5041 cgaggcactg gatgggtcct tcactaccga caccgtcttc cgcgtcaccg ccaccgggga
     5101 cggcttcacc ctgcgggagg tgccggtgga cccgcccctg accaaggtgt tccccgacga
     5161 cgaatcggac gacgaatcgg acgacgggga ggacggtgac ccggactctc ggacgttcgt
     5221 cgcgtacggg gacgacggcg acctggcggg cttcgtggtc gtctcgtact ccggctggaa
     5281 ccgccggctg accgtcgagg acatcgaggt cgccccggag caccgggggc acggggtcgg
     5341 gcgcgcgttg atggggctcg cgacggagtt cgcccgcgag cggggtgccg ggcacctctg
     5401 gctggaggtc accaacgtca acgcaccggc gatccacgcg taccggcgga tggggttcac
     5461 cctctgcggc ctggacaccg ccctgtacga cggcaccgcc tcggacggcg agcaggcgct
     5521 ctacatgagc atgccctgcc cctaagcact tcgtggccga ggagcaggac tgacacgttc
     5581 agaattggtt aattggttgt aacattattc agattgggcc ccgttccact gagcgtcaga
     5641 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     5701 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     5761 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     5821 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     5881 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     5941 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     6001 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     6061 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     6121 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     6181 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     6241 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     6301 gccttttgct cacatgtt
//
