LOCUS       sequence-12658-e        5123 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 5123)
  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 5123)
  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: pDRAV-4
FEATURES             Location/Qualifiers
     source          1..5123
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     623..642
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        623..641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(656..689)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    750..783
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        811..1310
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(846..869)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1243..1262
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             1329..2354
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             1329..2354
                     /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"
     protein_bind    2838..2871
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(2878..2911)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     promoter        complement(2935..2953)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2974..2990)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2974..2990)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2987..3009)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2998..3014
                     /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)."
     promoter        complement(join(3022..3028,3029..3046,3047..3052))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3067..3088
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3205..3222)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3376..3964)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3456..3475)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4135..4926,4927..4995))
                     /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     4758..4777
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4996..5100)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg agctcataac
      661 ttcgtatagc atacattata cgaagttatg gcgcctcagc ggccgcacaa tgcatgtagt
      721 taacttctta attaactggg atcctgcggg tgccagggcg tgcccttggg ctccccgggc
      781 gcgtactcca cctcacctcg agattctacc gggtagggga ggcgcttttc ccaaggcagt
      841 ctggagcatg cgctttagca gccccgctgg gcacttggcg ctacacaagt ggcctctggc
      901 ctcgcacaca ttccacatcc accggtaggc gccaaccggc tccgttcttt ggtggcccct
      961 tcgcgccacc ttctactcct cccctagtca ggaagttccc ccccgccccg cagctcgcgt
     1021 cgtgcaggac gtgacaaatg gaagtagcac gtctcactag tctcgtgcag atggacagca
     1081 ccgctgagca atggaagcgg gtaggccttt ggggcagcgg ccaatagcag ctttgctcct
     1141 tcgctttctg ggctcagagg ctgggaaggg gtgggtccgg gggcgggctc aggggcgggc
     1201 tcaggggcgg ggcgggcgcc cgaaggtcct ccggaggccc ggcattctgc acgcttcaaa
     1261 agcgcacgtc tgccgcgctg ttctcctctt cctcatctcc gggcctttcg accgatccag
     1321 ccgccaccat gaaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa
     1381 agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca
     1441 gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct
     1501 acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc
     1561 ttgacattgg ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg
     1621 tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg
     1681 ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac
     1741 cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc
     1801 atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc
     1861 tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg
     1921 atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga
     1981 gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt
     2041 ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag
     2101 gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct
     2161 tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc
     2221 gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga
     2281 ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga
     2341 gggcaaagga atagtcgaga aattgatgat ctattaaaca ataaagatgt ccactaaaat
     2401 ggaagttttt cctgtcatac tttgttaaga agggtgagaa cagagtacct acattttgaa
     2461 tggaaggatt ggagctacgg gggtgggggt ggggtgggat tagataaatg cctgctcttt
     2521 actgaaggct ctttactatt gctttatgat aatgtttcat agttggatat cataatttaa
     2581 acaagcaaaa ccaaattaag ggccagctca ttcctcccac tcatgatcta tagatctctc
     2641 gtgggatcat tgtttttctc ttgattccca ctttgtggtt ctaagtactg tggtttccaa
     2701 atgtgtcagt ttcatagcct gaagaacgag atcagcagcc tctgttccac atacacttca
     2761 ttctcagtat tgttacgcgt gtgccccaac tggggtaacc tttgagttct ctcagttggg
     2821 ggcgtagggt cgtcgacata acttcgtata aagtatccta tacgaagtta tatcgatgaa
     2881 gttcctatac tttctagaga ataggaactt caagcttggt accagctttt gttcccttta
     2941 gtgagggtta atttcgagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     3001 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     3061 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     3121 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     3181 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     3241 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     3301 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     3361 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     3421 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     3481 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     3541 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     3601 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     3661 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     3721 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     3781 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
     3841 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     3901 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     3961 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     4021 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     4081 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     4141 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     4201 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     4261 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     4321 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     4381 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     4441 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     4501 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     4561 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     4621 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     4681 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     4741 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     4801 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     4861 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     4921 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     4981 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     5041 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     5101 cacatttccc cgaaaagtgc cac
//
