LOCUS       sequence-274616-        4894 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Provides two loxP sequences between gene cassettes in the 
            destination vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4894)
  TITLE     Minoru Ko Lab plasmids
REFERENCE   2  (bases 1 to 4894)
  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"
COMMENT     Sequence Label: pDEST-flox-R4R3
FEATURES             Location/Qualifiers
     source          1..4894
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    576..710
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(613..632)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     667..686
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(718..751)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     764..780
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     764..780
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    800..923
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(1271..1576)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(1373..1392)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1896..2579)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     2494..2513
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2580..2682)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(2828..2951)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2959..2975)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2988..3021)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(3087..3104)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3258..3846)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3338..3357)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4017..4808,4809..4877))
                     /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     4640..4659
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gaccgctttg gaaaaacaaa gactgtattt cctggaaatt aatgtttatt caataaactg
       61 tgtattcagc tatattcaca tagtggtgag gctgaaatga ggcgggaaga ggcggttggg
      121 gcttaattat atcaatttgg gtggccccac agcgcctcca aggcgccagt cctgttttga
      181 caagttgcct ctggaagcct ctcttctttt tctccagagt aagcggaggc caggggcccc
      241 cggcctctgc ttaatactaa aaaaaacagc tgttgtcata gtaatgattg ggtggaaaca
      301 ttccaggcct gggtggagag gctttttgct tcctcttgca aaaccacact gacattccag
      361 gcctgggtgg agaggctttt tgcttcctct tgcaaaacca cactgccctc tggagggcag
      421 ttgcctagca actaattaaa agaggatgtc gcacggccag ctgcggtcag ttagtcactt
      481 cctgcttaac tgacttgaca ttttctattt taagagtcgg gaggaaaatt actgtgttgg
      541 aggccctccg ccatcttctg aagctgaatc gaattaactt gtttattgca gcttataatg
      601 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
      661 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc tgatatcata
      721 acttcgtata gcatacatta tacgaagtta tgtcgacact agtcaggaaa cagctatgac
      781 catgattacg ccaagctatc aactttgtat agaaaagttg aacgagaaac gtaaaatgat
      841 ataaatatca atatattaaa ttagattttg cataaaaaac agactacata atactgtaaa
      901 acacaacata tccagtcact atggtcgacc gacagccttc caaatgttct tctcaaacgg
      961 aatcgtcgta tccagcctac tcgctattgt cctcaatgcc gtattaaatc ataaaaagaa
     1021 ataagaaaaa gaggtgcgag cctctttttt gtgtgacaaa ataaaaacat ctacctattc
     1081 atatacgcta gtgtcatagt cctgaaaatc atctgcatca agaacaattt cacaactctt
     1141 atacttttct cttacaagtc gttcggcttc atctggattt tcagcctcta tacttactaa
     1201 acgtgataaa gtttctgtaa tttctactgt atcgacctgc agactggctg tgtataaggg
     1261 agcctgacat ttatattccc cagaacatca ggttaatggc gtttttgatg tcattttcgc
     1321 ggtggctgag atcagccact tcttccccga taacggagac cggcacactg gccatatcgg
     1381 tggtcatcat gcgccagctt tcatccccga tatgcaccac cgggtaaagt tcacgggaga
     1441 ctttatctga cagcagacgt gcactggcca gggggatcac catccgtcgc ccgggcgtgt
     1501 caataatatc actctgtaca tccacaaaca gacgataacg gctctctctt ttataggtgt
     1561 aaaccttaaa ctgcatttca ccagcccctg ttctcgtcag caaaagagcc gttcatttca
     1621 ataaaccggg cgacctcagc catcccttcc tgattttccg ctttccagcg ttcggcacgc
     1681 agacgacggg cttcattctg catggttgtg cttaccagac cggagatatt gacatcatat
     1741 atgccttgag caactgatag ctgtcgctgt caactgtcac tgtaatacgc tgcttcatag
     1801 catacctctt tttgacatac ttcgggtata catatcagta tatattctta taccgcaaaa
     1861 atcagcgcgc aaatacgcat actgttatct ggcttttagt aagccggatc cacgcggcgt
     1921 ttacgccccc cctgccactc atcgcagtac tgttgtaatt cattaagcat tctgccgaca
     1981 tggaagccat cacaaacggc atgatgaacc tgaatcgcca gcggcatcag caccttgtcg
     2041 ccttgcgtat aatatttgcc catggtgaaa acgggggcga agaagttgtc catattggcc
     2101 acgtttaaat caaaactggt gaaactcacc cagggattgg ctgagacgaa aaacatattc
     2161 tcaataaacc ctttagggaa ataggccagg ttttcaccgt aacacgccac atcttgcgaa
     2221 tatatgtgta gaaactgccg gaaatcgtcg tggtattcac tccagagcga tgaaaacgtt
     2281 tcagtttgct catggaaaac ggtgtaacaa gggtgaacac tatcccatat caccagctca
     2341 ccgtctttca ttgccatacg gaattccgga tgagcattca tcaggcgggc aagaatgtga
     2401 ataaaggccg gataaaactt gtgcttattt ttctttacgg tctttaaaaa ggccgtaata
     2461 tccagctgaa cggtctggtt ataggtacat tgagcaactg actgaaatgc ctcaaaatgt
     2521 tctttacgat gccattggga tatatcaacg gtggtatatc cagtgatttt tttctccatt
     2581 ttagcttcct tagctcctga aaatctcgat aactcaaaaa atacgcccgg tagtgatctt
     2641 atttcattat ggtgaaagtt ggaacctctt acgtgccgat caacgtctca ttttcgccaa
     2701 aagttggccc agggcttccc ggtatcaaca gggacaccag gatttattta ttctgcgaag
     2761 tgatcttccg tcacaggtat ttattcggcg caaagtgcgt cgggtgatgc tgccaactta
     2821 cggccgccat agtgactgga tatgttgtgt tttacagtat tatgtagtct gttttttatg
     2881 caaaatctaa tttaatatat tgatatttat atcattttac gtttctcgtt caactttatt
     2941 atacatagtt gataattcac tggccgtcgt tttacctcga gggatccata acttcgtata
     3001 gcatacatta tacgaagtta tgctgcataa tgaatcggcc aacgcgcggg gagaggcggt
     3061 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3121 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     3181 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     3241 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     3301 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     3361 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     3421 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     3481 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     3541 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     3601 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     3661 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     3721 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     3781 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     3841 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     3901 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     3961 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4021 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     4081 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     4141 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     4201 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     4261 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     4321 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     4381 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     4441 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     4501 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     4561 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     4621 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     4681 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     4741 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     4801 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     4861 aaatgttgaa tactcatact cttccttttt caat
//
