LOCUS       sequence-12654-e        7669 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 7669)
  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 7669)
  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: pDIRE
FEATURES             Location/Qualifiers
     source          1..7669
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        24..128
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(129..197,198..989)
                     /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     complement(347..366)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1160..1748
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1649..1668
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1902..1919
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2036..2057
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2072..2077,2078..2095,2096..2102)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2110..2126
                     /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     2115..2137
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2134..2150
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2134..2150
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2169..2189
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2171..2189
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     2217..2233
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2218..2234
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2259..2758
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2294..2317)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2691..2710
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      2772..2781
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2778..2783,2784..2804,2805..4076)
                     /codon_start=1
                     /product="nuclear-targeted site-specific recombinase"
                     /label=FLPo
                     /note="FLPo is a mouse codon-optimized fusion between the 
                     SV40 nuclear localization signal and a thermostable version
                     of the Saccharomyces cerevisiae site-specific recombinase 
                     FLP.  Recombination occurs at FRT sequences."
                     /translation="MAPKKKRKVMSQFDILCKTPPKVLVRQFVERFERPSGEKIASCAA
                     ELTYLCWMITHNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKK
                     LIPAWEFTIIPYNGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESI
                     WEITEKILNSFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGV
                     IIQCLVTETKTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEY
                     QLLKDNLVRSYNKALKKNAPYPIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWS
                     DKRASAVARTTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQL
                     KGSAEGSIRYPAWNGIISQEVLDYLSSYINRRI"
     primer_bind     complement(4104..4121)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4110..4334
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4428..4447)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4429..4447)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4457..4474)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4457..4473)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4466..4488)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        4800..5978
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          5031..5969
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     5926..5946
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      5995..6004
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6007..6027
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    7176..7257
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7213..7232)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7267..7286
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(7308..7666)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7348..7369)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7560..7579
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
       61 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
      121 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
      181 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
      241 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
      301 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
      361 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
      421 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
      481 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
      541 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
      601 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
      661 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
      721 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
      781 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
      841 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
      901 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
      961 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     1021 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     1081 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     1141 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     1201 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     1261 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     1321 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     1381 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     1441 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     1501 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     1561 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     1621 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     1681 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     1741 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     1801 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     1861 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     1921 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     1981 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     2041 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     2101 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     2161 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggtaccgggc cccccctcga
     2221 ggtcgacggt atcgataagc ttgatatcga attctaccgg gtaggggagg cgcttttccc
     2281 aaggcagtct ggagcatgcg ctttagcagc cccgctgggc acttggcgct acacaagtgg
     2341 cctctggcct cgcacacatt ccacatccac cggtaggcgc caaccggctc cgttctttgg
     2401 tggccccttc gcgccacctt ctactcctcc cctagtcagg aagttccccc ccgccccgca
     2461 gctcgcgtcg tgcaggacgt gacaaatgga agtagcacgt ctcactagtc tcgtgcagat
     2521 ggacagcacc gctgagcaat ggaagcgggt aggcctttgg ggcagcggcc aatagcagct
     2581 ttgctccttc gctttctggg ctcagaggct gggaaggggt gggtccgggg gcgggctcag
     2641 gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc ggaggcccgg cattctgcac
     2701 gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc tcatctccgg gcctttcgac
     2761 ctgcaatcgc cgccaccatg gctcctaaga agaagaggaa ggtgatgagc cagttcgaca
     2821 tcctgtgcaa gacccccccc aaggtgctgg tgcggcagtt cgtggagaga ttcgagaggc
     2881 ccagcggcga gaagatcgcc agctgtgccg ccgagctgac ctacctgtgc tggatgatca
     2941 cccacaacgg caccgccatc aagagggcca ccttcatgag ctacaacacc atcatcagca
     3001 acagcctgag cttcgacatc gtgaacaaga gcctgcagtt caagtacaag acccagaagg
     3061 ccaccatcct ggaggccagc ctgaagaagc tgatccccgc ctgggagttc accatcatcc
     3121 cttacaacgg ccagaagcac cagagcgaca tcaccgacat cgtgtccagc ctgcagctgc
     3181 agttcgagag cagcgaggag gccgacaagg gcaacagcca cagcaagaag atgctgaagg
     3241 ccctgctgtc cgagggcgag agcatctggg agatcaccga gaagatcctg aacagcttcg
     3301 agtacaccag caggttcacc aagaccaaga ccctgtacca gttcctgttc ctggccacat
     3361 tcatcaactg cggcaggttc agcgacatca agaacgtgga ccccaagagc ttcaagctgg
     3421 tgcagaacaa gtacctgggc gtgatcattc agtgcctggt gaccgagacc aagacaagcg
     3481 tgtccaggca catctacttt ttcagcgcca gaggcaggat cgaccccctg gtgtacctgg
     3541 acgagttcct gaggaacagc gagcccgtgc tgaagagagt gaacaggacc ggcaacagca
     3601 gcagcaacaa gcaggagtac cagctgctga aggacaacct ggtgcgcagc tacaacaagg
     3661 ccctgaagaa gaacgccccc taccccatct tcgctatcaa gaacggccct aagagccaca
     3721 tcggcaggca cctgatgacc agctttctga gcatgaaggg cctgaccgag ctgacaaacg
     3781 tggtgggcaa ctggagcgac aagagggcct ccgccgtggc caggaccacc tacacccacc
     3841 agatcaccgc catccccgac cactacttcg ccctggtgtc caggtactac gcctacgacc
     3901 ccatcagcaa ggagatgatc gccctgaagg acgagaccaa ccccatcgag gagtggcagc
     3961 acatcgagca gctgaagggc agcgccgagg gcagcatcag ataccccgcc tggaacggca
     4021 tcatcagcca ggaggtgctg gactacctga gcagctacat caacaggcgg atctgatgag
     4081 atatcctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     4141 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     4201 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     4261 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     4321 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctcgagat ccactagttc
     4381 tagcctcgag gctagagcgg ccgccaccgc ggtggagctc caattcgccc tatagtgagt
     4441 cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
     4501 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
     4561 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggaaattgt
     4621 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     4681 ccaataggcc gaaatcggca aaatccctta atcaagcttt gcaaagatgg ataaagtttt
     4741 aaacagagag gaatctttgc agctaatgga ccttctaggt cttgaaagga gtgggaattg
     4801 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg
     4861 gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg
     4921 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag
     4981 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg
     5041 tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta
     5101 cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg
     5161 agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc
     5221 ttgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt
     5281 tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc
     5341 aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg
     5401 cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag
     5461 cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg
     5521 gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag
     5581 ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga
     5641 cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt
     5701 cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt
     5761 agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg
     5821 agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat
     5881 tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag
     5941 tggttcaaag tttttttctt ccatttcagg tgtcgtgaag gaattggatc cgccgccacc
     6001 atggtgccca agaagaagag gaaagtctcc aacctgctga ctgtgcacca aaacctgcct
     6061 gccctccctg tggatgccac ctctgatgaa gtcaggaaga acctgatgga catgttcagg
     6121 gacaggcagg ccttctctga acacacctgg aagatgctcc tgtctgtgtg cagatcctgg
     6181 gctgcctggt gcaagctgaa caacaggaaa tggttccctg ctgaacctga ggatgtgagg
     6241 gactacctcc tgtacctgca agccagaggc ctggctgtga agaccatcca acagcacctg
     6301 ggccagctca acatgctgca caggagatct ggcctgcctc gcccttctga ctccaatgct
     6361 gtgtccctgg tgatgaggag aatcagaaag gagaatgtgg atgctgggga gagagccaag
     6421 caggccctgg cctttgaacg cactgacttt gaccaagtca gatccctgat ggagaactct
     6481 gacagatgcc aggacatcag gaacctggcc ttcctgggca ttgcctacaa caccctgctg
     6541 cgcattgccg aaattgccag aatcagagtg aaggacatct cccgcaccga tggtgggaga
     6601 atgctgatcc acattggcag gaccaagacc ctggtgtcca cagctggtgt ggagaaggcc
     6661 ctgtccctgg gggttaccaa gctggtggag agatggatct ctgtgtctgg tgtggctgat
     6721 gaccccaaca actacctgtt ctgccgggtc agaaagaatg gtgtggctgc cccttctgcc
     6781 acctcccaac tgtccacccg ggccctggaa gggatctttg aggccaccca ccgcctgatc
     6841 tatggtgcca aggatgactc tgggcagaga tacctggcct ggtctggcca ctctgccaga
     6901 gtgggtgctg ccagggacat ggccagggct ggtgtgtcca tccctgaaat catgcaggct
     6961 ggtggctgga ccaatgtgaa cattgtgatg aactacatca gaaacctgga ctctgagact
     7021 ggggccatgg tgaggctgct cgaggatggg gactgaacta gtgactctag atcataatca
     7081 gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga
     7141 acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg
     7201 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     7261 ctagttgtgg tttgtccaaa ctcatcaatg tatactagta tagagattaa atcaaaagaa
     7321 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     7381 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     7441 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     7501 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     7561 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     7621 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcag
//
