LOCUS       sequence-12660-e        6332 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 6332)
  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 6332)
  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-1
FEATURES             Location/Qualifiers
     source          1..6332
                     /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             1449..1502
                     /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             1509..1883
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="PEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSIYVYKVL
                     KQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPG
                     ELAKHAVSEGTKAVTKYTSAK"
     CDS             join(1890..1892,1893..1895,1896..2609)
                     /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(1935..1956)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2196..2215)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2543..2564
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2732..2866
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2769..2788)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2823..2842
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    2876..2907
                     /label=rox
                     /bound_moiety="Dre recombinase"
     promoter        2949..3448
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2984..3007)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3381..3400
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3488..4087
                     /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(3488..3507)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3984..4004
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    4628..4659
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    complement(4668..4701)
                     /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(4723..4822)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        4921..4968
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4987..5559
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     rep_origin      5682..6270
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6171..6190
                     /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 cgcagatcgc
     1441 agatctccga gggcagagga agtcttctaa catgcggtga cgtggaggag aatcccggac
     1501 cggggggacc agagccagcg aagtctgctc ccgccccgaa aaagggctcc aagaaggcgg
     1561 tgactaaggc gcagaagaaa gacggcaaga agcgcaagcg cagccgcaag gagagctatt
     1621 ccatctatgt gtacaaggtt ctgaagcagg tccaccctga caccggcatt tcgtccaagg
     1681 ccatgggcat catgaattcg tttgtgaacg acattttcga gcgcatcgca ggtgaggctt
     1741 cccgcctggc gcattacaac aagcgctcga ccatcacctc cagggagatc cagacggccg
     1801 tgcgcctgct gctgcctggg gagttggcca agcacgccgt gtccgagggt actaaggccg
     1861 tcaccaagta caccagcgct aaatccacca tggtgagcaa gggcgaggag ctgttcaccg
     1921 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     1981 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagctg atctgcacca
     2041 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgggctac ggcctgcagt
     2101 gcttcgcccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     2161 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     2221 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     2281 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     2341 tctatatcac cgccgacaag cagaagaacg gcatcaaggc caacttcaag atccgccaca
     2401 acatcgagga cggcggcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     2461 acggccccgt gctgctgccc gacaaccact acctgagcta ccagtccgcc ctgagcaaag
     2521 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     2581 ctctcggcat ggacgagctg tacaagtaag aagagcttaa ttaatgatca taatcagcca
     2641 tatcacatct gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct
     2701 gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta
     2761 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     2821 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatccgtt taaactaact
     2881 ttaaataatt ggcattattt aaagttacat atggcggccg ccctaaggct cgacctcgaa
     2941 attctaccgg gtaggggagg cgcttttccc aaggcagtct ggagcatgcg ctttagcagc
     3001 cccgctgggc acttggcgct acacaagtgg cctctggcct cgcacacatt ccacatccac
     3061 cggtaggcgc caaccggctc cgttctttgg tggccccttc gcgccacctt ctactcctcc
     3121 cctagtcagg aagttccccc ccgccccgca gctcgcgtcg tgcaggacgt gacaaatgga
     3181 agtagcacgt ctcactagtc tcgtgcagat ggacagcacc gctgagcaat ggaagcgggt
     3241 aggcctttgg ggcagcggcc aatagcagct ttgctccttc gctttctggg ctcagaggct
     3301 gggaaggggt gggtccgggg gcgggctcag gggcgggctc aggggcgggg cgggcgcccg
     3361 aaggtcttcc ggaggcccgg cattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt
     3421 ctcctcttcc tcatctccgg gcctttcgac ctgcatccat ctagatctcg agcagctgaa
     3481 gcttaccatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag
     3541 ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga
     3601 tccagatcgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg
     3661 gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac
     3721 gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt
     3781 gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc
     3841 caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg
     3901 tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc
     3961 cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt
     4021 caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg
     4081 tgcctgacgc ccgccccacg acccgcagcg cccgaccgaa aggagcgcac gaccccatgc
     4141 atcgatgata tcagatcccc gggatgcaga aattgatgat ctattaaaca ataaagatgt
     4201 ccactaaaat ggaagttttt cctgtcatac tttgttaaga agggtgagaa cagagtacct
     4261 acattttgaa tggaaggatt ggagctacgg gggtgggggt ggggtgggat tagataaatg
     4321 cntgctcttt actgaaggct ctttactatt gctttatgat aatgtttcat agttggatat
     4381 cataatttaa acaagcaaaa ccaaattaag ggccagctca ttcctcccac tcatgatcta
     4441 tagatctata gatctctcgt gggatcattg tttttctctt gattcccact ttgtggttct
     4501 aagtactgtg gtttccaaat gtgtcagttt catagcctga agaacgagat cagcagcctc
     4561 tgttccacat acacttcatt ctcagtattg ttttgccaag ttctaattcc atcagaagct
     4621 ggtcgagtaa ctttaaataa ttggcattat ttaaagttag gcgcgccata acttcgtata
     4681 gcatacatta tacgaagtta tgtcgagata tttaaatatt agacccagct ttcttgtaca
     4741 aagttggcat tataagaaag cattgcttat caatttgttg caacgaacag gtcactatca
     4801 gtcaaaataa aatcattatt tgccatccag ctgcagctct ggcccgtgtc tcaaaatctc
     4861 tgatgttaca ttgcacaaga taaaagcttg tatatccatt ttcggatctg atcagcacgt
     4921 gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac
     4981 taaaccatgg gtaccactct tgacgacacg gcttaccggt accgcaccag tgtcccgggg
     5041 gacgccgagg ccatcgaggc actggatggg tccttcacta ccgacaccgt cttccgcgtc
     5101 accgccaccg gggacggctt caccctgcgg gaggtgccgg tggacccgcc cctgaccaag
     5161 gtgttccccg acgacgaatc ggacgacgaa tcggacgacg gggaggacgg tgacccggac
     5221 tctcggacgt tcgtcgcgta cggggacgac ggcgacctgg cgggcttcgt ggtcgtctcg
     5281 tactccggct ggaaccgccg gctgaccgtc gaggacatcg aggtcgcccc ggagcaccgg
     5341 gggcacgggg tcgggcgcgc gttgatgggg ctcgcgacgg agttcgcccg cgagcggggt
     5401 gccgggcacc tctggctgga ggtcaccaac gtcaacgcac cggcgatcca cgcgtaccgg
     5461 cggatggggt tcaccctctg cggcctggac accgccctgt acgacggcac cgcctcggac
     5521 ggcgagcagg cgctctacat gagcatgccc tgcccctaag cacttcgtgg ccgaggagca
     5581 ggactgacac gttcagaatt ggttaattgg ttgtaacatt attcagattg ggccccgttc
     5641 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     5701 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     5761 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     5821 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     5881 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     5941 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     6001 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     6061 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     6121 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     6181 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     6241 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     6301 ctggcctttt gctggccttt tgctcacatg tt
//
