LOCUS       sequence-12661-e        6325 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 6325)
  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 6325)
  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-2
FEATURES             Location/Qualifiers
     source          1..6325
                     /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             1442..1495
                     /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             1502..1876
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="PEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSIYVYKVL
                     KQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPG
                     ELAKHAVSEGTKAVTKYTSAK"
     CDS             join(1883..1885,1886..1888,1889..2602)
                     /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(1928..1949)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2189..2208)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2536..2557
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2725..2859
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2762..2781)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2816..2835
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    2869..2900
                     /label=rox
                     /bound_moiety="Dre recombinase"
     promoter        2942..3441
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2977..3000)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3374..3393
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3481..4080
                     /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(3481..3500)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3977..3997
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    4621..4652
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    complement(4661..4694)
                     /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(4716..4815)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        4914..4961
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4980..5552
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     rep_origin      5675..6263
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6164..6183
                     /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 cgcagatctc
     1441 cgagggcaga ggaagtcttc taacatgcgg tgacgtggag gagaatcccg gaccgggggg
     1501 accagagcca gcgaagtctg ctcccgcccc gaaaaagggc tccaagaagg cggtgactaa
     1561 ggcgcagaag aaagacggca agaagcgcaa gcgcagccgc aaggagagct attccatcta
     1621 tgtgtacaag gttctgaagc aggtccaccc tgacaccggc atttcgtcca aggccatggg
     1681 catcatgaat tcgtttgtga acgacatttt cgagcgcatc gcaggtgagg cttcccgcct
     1741 ggcgcattac aacaagcgct cgaccatcac ctccagggag atccagacgg ccgtgcgcct
     1801 gctgctgcct ggggagttgg ccaagcacgc cgtgtccgag ggtactaagg ccgtcaccaa
     1861 gtacaccagc gctaaatcca ccatggtgag caagggcgag gagctgttca ccggggtggt
     1921 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     1981 gggcgagggc gatgccacct acggcaagct gaccctgaag ctgatctgca ccaccggcaa
     2041 gctgcccgtg ccctggccca ccctcgtgac caccctgggc tacggcctgc agtgcttcgc
     2101 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     2161 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     2221 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     2281 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     2341 caccgccgac aagcagaaga acggcatcaa ggccaacttc aagatccgcc acaacatcga
     2401 ggacggcggc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     2461 cgtgctgctg cccgacaacc actacctgag ctaccagtcc gccctgagca aagaccccaa
     2521 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     2581 catggacgag ctgtacaagt aagaagagct taattaatga tcataatcag ccatatcaca
     2641 tctgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat
     2701 aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa
     2761 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     2821 ttgtccaaac tcatcaatgt atcttatcat gtctggatcc gtttaaacta actttaaata
     2881 attggcatta tttaaagtta catatggcgg ccgccctaag gctcgacctc gaaattctac
     2941 cgggtagggg aggcgctttt cccaaggcag tctggagcat gcgctttagc agccccgctg
     3001 ggcacttggc gctacacaag tggcctctgg cctcgcacac attccacatc caccggtagg
     3061 cgccaaccgg ctccgttctt tggtggcccc ttcgcgccac cttctactcc tcccctagtc
     3121 aggaagttcc cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca
     3181 cgtctcacta gtctcgtgca gatggacagc accgctgagc aatggaagcg ggtaggcctt
     3241 tggggcagcg gccaatagca gctttgctcc ttcgctttct gggctcagag gctgggaagg
     3301 ggtgggtccg ggggcgggct caggggcggg ctcaggggcg gggcgggcgc ccgaaggtct
     3361 tccggaggcc cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct
     3421 tcctcatctc cgggcctttc gacctgcatc catctagatc tcgagcagct gaagcttacc
     3481 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta
     3541 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccagat
     3601 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac
     3661 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag
     3721 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt
     3781 tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag
     3841 cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc
     3901 agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg
     3961 gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc
     4021 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga
     4081 cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg cacgacccca tgcatcgatg
     4141 atatcagatc cccgggatgc agaaattgat gatctattaa acaataaaga tgtccactaa
     4201 aatggaagtt tttcctgtca tactttgtta agaagggtga gaacagagta cctacatttt
     4261 gaatggaagg attggagcta cgggggtggg ggtggggtgg gattagataa atgcntgctc
     4321 tttactgaag gctctttact attgctttat gataatgttt catagttgga tatcataatt
     4381 taaacaagca aaaccaaatt aagggccagc tcattcctcc cactcatgat ctatagatct
     4441 atagatctct cgtgggatca ttgtttttct cttgattccc actttgtggt tctaagtact
     4501 gtggtttcca aatgtgtcag tttcatagcc tgaagaacga gatcagcagc ctctgttcca
     4561 catacacttc attctcagta ttgttttgcc aagttctaat tccatcagaa gctggtcgag
     4621 taactttaaa taattggcat tatttaaagt taggcgcgcc ataacttcgt atagcataca
     4681 ttatacgaag ttatgtcgag atatttaaat attagaccca gctttcttgt acaaagttgg
     4741 cattataaga aagcattgct tatcaatttg ttgcaacgaa caggtcacta tcagtcaaaa
     4801 taaaatcatt atttgccatc cagctgcagc tctggcccgt gtctcaaaat ctctgatgtt
     4861 acattgcaca agataaaagc ttgtatatcc attttcggat ctgatcagca cgtgttgaca
     4921 attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca
     4981 tgggtaccac tcttgacgac acggcttacc ggtaccgcac cagtgtcccg ggggacgccg
     5041 aggccatcga ggcactggat gggtccttca ctaccgacac cgtcttccgc gtcaccgcca
     5101 ccggggacgg cttcaccctg cgggaggtgc cggtggaccc gcccctgacc aaggtgttcc
     5161 ccgacgacga atcggacgac gaatcggacg acggggagga cggtgacccg gactctcgga
     5221 cgttcgtcgc gtacggggac gacggcgacc tggcgggctt cgtggtcgtc tcgtactccg
     5281 gctggaaccg ccggctgacc gtcgaggaca tcgaggtcgc cccggagcac cgggggcacg
     5341 gggtcgggcg cgcgttgatg gggctcgcga cggagttcgc ccgcgagcgg ggtgccgggc
     5401 acctctggct ggaggtcacc aacgtcaacg caccggcgat ccacgcgtac cggcggatgg
     5461 ggttcaccct ctgcggcctg gacaccgccc tgtacgacgg caccgcctcg gacggcgagc
     5521 aggcgctcta catgagcatg ccctgcccct aagcacttcg tggccgagga gcaggactga
     5581 cacgttcaga attggttaat tggttgtaac attattcaga ttgggccccg ttccactgag
     5641 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     5701 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     5761 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     5821 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     5881 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     5941 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     6001 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     6061 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     6121 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     6181 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     6241 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     6301 tttgctggcc ttttgctcac atgtt
//
