LOCUS       sequence-382236-        7728 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of FLPe recombinase from the highly-expressed 
            CAGGS promoter; puro selectable marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7728)
  AUTHORS   Beard C, Hochedlinger K, Plath K, Wutz A, Jaenisch R
  TITLE     Efficient method to generate single-copy transgenic mice by 
            site-specific integration in embryonic stem cells.
  JOURNAL   Genesis. 2006 Jan . 44(1):23-8.
  PUBMED    16400644
REFERENCE   2  (bases 1 to 7728)
  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: "Genesis. 
            2006 Jan . 44(1):23-8."
COMMENT     Sequence Label: pCAGGS-flpE-puro
FEATURES             Location/Qualifiers
     source          1..7728
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    514..648
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        662..1041
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          1319..2335
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2259..2281
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2343..2362
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2396..2405
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2402..2407,2408..2428,2429..3700)
                     /codon_start=1
                     /product="nuclear-targeted site-specific recombinase"
                     /label=FLPe
                     /note="FLPe is a fusion between the SV40 nuclear 
                     localization signal and a thermostable version of the 
                     Saccharomyces cerevisiae site-specific recombinase FLP. 
                     Recombination occurs at FRT sequences (Buchholz et al., 
                     1998)."
                     /translation="MAPKKKRKVMSQFDILCKTPPKVLVRQFVERFERPSGEKIASCAA
                     ELTYLCWMITHNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKK
                     LIPAWEFTIIPYNGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESI
                     WEITEKILNSFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGV
                     IIQCLVTETKTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEY
                     QLLKDNLVRSYNKALKKNAPYPIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWS
                     DKRASAVARTTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQL
                     KGSAEGSIRYPAWNGIISQEVLDYLSSYINRRI"
     misc_feature    join(3739..4313,4314..4316,4317..4325)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3906..3923)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4133..4152
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4326..4925
                     /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(4326..4345)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4822..4842
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5006..5023)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5012..5236
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5283..5299)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5283..5299)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5296..5318)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5307..5323
                     /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(5331..5337,5338..5355,5356..5361))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5376..5397
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5456..5651
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5502..5637
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5657..5791
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5859..5876)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6030..6618)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6110..6129)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6789..7580,7581..7649))
                     /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     7412..7431
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tattcagcta tattcacata gtggtgaggc tgaaatgagg cgggaagagg cggttggggc
       61 ttaattatat caatttgggt ggccccacag cgcctccaag gcgccagtcc tgttttgaca
      121 agttgcctct ggaagcctct cttctttttc tccagagtaa gcggaggcca ggggcccccg
      181 gcctctgctt aatactaaaa aaaacagctg ttgtcatagt aatgattggg tggaaacatt
      241 ccaggcctgg gtggagaggc tttttgcttc ctcttgcaaa accacactga cattccaggc
      301 ctgggtggag aggctttttg cttcctcttg caaaaccaca ctgccctctg gagggcagtt
      361 gcctagcaac taattaaaag aggatgtcgc acggccagct gcggtcagtt agtcacttcc
      421 tgcttaactg acttgacatt ttctatttta agagtcggga ggaaaattac tgtgttggag
      481 gccctccgcc atcttctgaa gctgaatcga attaacttgt ttattgcagc ttataatggt
      541 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
      601 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatctg atatcatcgt
      661 cgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc
      721 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
      781 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
      841 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
      901 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
      961 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
     1021 ttagtcatcg ctattaccat ggtcgaggtg agccccacgt tctgcttcac tctccccatc
     1081 tcccccccct ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg
     1141 atgggggcgg gggggggggg ggggcgcgcc rggggggggg gggggggggg gggggggggg
     1201 gggggsgrgg sggrgrggkg sggcggcagc caatcagagc ggcgcgctcc gaaagtttcc
     1261 ttttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg
     1321 agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc
     1381 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
     1441 gggctgtaat tagcgcttgg tttaatgacg gcttgtttct tttctgtggc tgcgtgaaag
     1501 ccttgagggg ctccgggagg gccctttgtg cggggggagc ggctcggggg gtgcgtgcgt
     1561 gtgtgtgtgc gtggggagcg ccgcgtgcgg ctccgcgctg cccggcggct gtgagcgctg
     1621 cgggcgcggc gcggggcttt gtgcgctccg cagtgtgcgc gaggggagcg cggccggggg
     1681 cggtgccccg cggtgcgggg ggggctgcga ggggaacaaa ggctgcgtgc ggggtgtgtg
     1741 cgtggggggg tgagcagggg gtgtgggcgc gtcggtcggg ctgcaacccc ccctgcaccc
     1801 ccctccccga gttgctgagc acggcccggc ttcgggtgcg gggctccgta cggggcgtgg
     1861 cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg ggggtgccgg gcggggcggg
     1921 gccgcckcgg gccggggggg gctcgggggr ggggcgcggc ggcccccgga gcgccggcgg
     1981 ctgtcgaggc gcggcgagcc gcagccattg ccttttatgg taatcgtgcg agagggcgca
     2041 gggacttcct ttgtcccaaa tctgtgcgga gccgaaatct gggaggcgcc gccgcacccc
     2101 ctctagcggg cgcggggcga agcggtgcgg cgccggcagg aaggaaatgg gcggggaggg
     2161 ccttcgtgcg tcgccgcgcc gccgtcccct tctccctctc cagcctcggg gctgtccgcg
     2221 gggggacggc tgccttcggg ggggacgggg cagggcgggg ttcggcttct ggcgtgtgac
     2281 cggcggctct agagcctctg ctaaccatgt tcatgccttc ttctttttcc tacagctcct
     2341 gggcaacgtg ctggttattg tgctgtctca tcattttggc aaagaattac ccgccgccac
     2401 catggctccc aagaagaaga ggaaggtgat gagtcaattt gatatattat gtaaaacacc
     2461 acctaaggtc ctggttcgtc agtttgtgga aaggtttgaa agaccttcag gggaaaaaat
     2521 agcatcatgt gctgctgaac taacctattt atgttggatg attactcata acggaacagc
     2581 aatcaagaga gccacattca tgagctataa tactatcata agcaattcgc tgagtttcga
     2641 tattgtcaac aaatcactcc agtttaaata caagacgcaa aaagcaacaa ttctggaagc
     2701 ctcattaaag aaattaattc ctgcttggga atttacaatt attccttaca atggacaaaa
     2761 acatcaatct gatatcactg atattgtaag tagtttgcaa ttacagttcg aatcatcgga
     2821 agaagcagat aagggaaata gccacagtaa aaaaatgctt aaagcacttc taagtgaggg
     2881 tgaaagcatc tgggagatca ctgagaaaat actaaattcg tttgagtata cctcgagatt
     2941 tacaaaaaca aaaactttat accaattcct cttcctagct actttcatca attgtggaag
     3001 attcagcgat attaagaacg ttgatccgaa atcatttaaa ttagtccaaa ataagtatct
     3061 gggagtaata atccagtgtt tagtgacaga gacaaagaca agcgttagta ggcacatata
     3121 cttctttagc gcaaggggta ggatcgatcc acttgtatat ttggatgaat ttttgaggaa
     3181 ttctgaacca gtcctaaaac gagtaaatag gaccggcaat tcttcaagca acaaacagga
     3241 ataccaatta ttaaaagata acttagtcag atcgtacaac aaggctttga agaaaaatgc
     3301 gccttatcca atctttgcta taaagaatgg cccaaaatct cacattggaa gacatttgat
     3361 gacctcattt ctgtcaatga agggcctaac ggagttgact aatgttgtgg gaaattggag
     3421 cgataagcgt gcttctgccg tggccaggac aacgtatact catcagataa cagcaatacc
     3481 tgatcactac ttcgcactag tttctcggta ctatgcatat gatccaatat caaaggaaat
     3541 gatagcattg aaggatgaga ctaatccaat tgaggagtgg cagcatatag aacagctaaa
     3601 gggtagtgct gaaggaagca tacgataccc cgcatggaat gggataatat cacaggaggt
     3661 actagactac ctttcatcct acataaatag acgcatataa tgatctagag tcgagttaat
     3721 taagaattcc gccccccccc cctctccctc ccccccccct aacgttactg gccgaagccg
     3781 cttggaataa ggccggtgtg cgtttgtcta tatgttattt tccaccatat tgccgtcttt
     3841 tggcaatgtg agggcccgga aacctggccc tgtcttcttg acgagcattc ctaggggtct
     3901 ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct
     3961 ggaagcttct tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc ggaacccccc
     4021 acctggcgac aggtgcctct gcggccaaaa gccacgtgta taagatacac ctgcaaaggc
     4081 ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg gaaagagtca aatggctctc
     4141 ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag gtaccccatt gtatgggatc
     4201 tgatctgggg cctcggtgca catgctttac atgtgtttag tcgaggttaa aaaaacgtct
     4261 aggccccccg aaccacgggg acgtggtttt cctttgaaaa acacgatgat aatatggcca
     4321 caaccatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg
     4381 ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc
     4441 cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc
     4501 tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc
     4561 cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga
     4621 gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca
     4681 aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc
     4741 tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct
     4801 tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca
     4861 ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg
     4921 cctgacgccc gccccacgac ccgcagcgcc cgaccgaaag gagcgcacga ccccatgcat
     4981 cgatgatcta gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     5041 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     5101 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     5161 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     5221 gcggtgggct ctatggcttc tgaggcggaa agaacctgca gcccaagctt ggcgtaatca
     5281 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     5341 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     5401 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg gatccgcatc
     5461 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     5521 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     5581 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     5641 gcttttgcaa aaagctaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     5701 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     5761 actcatcaat gtatcttatc atgtctggat ccgctgcatt aatgaatcgg ccaacgcgcg
     5821 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     5881 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     5941 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     6001 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     6061 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     6121 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     6181 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     6241 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     6301 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     6361 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     6421 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     6481 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     6541 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     6601 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     6661 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     6721 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     6781 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     6841 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     6901 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     6961 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     7021 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     7081 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     7141 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     7201 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     7261 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     7321 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     7381 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     7441 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     7501 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     7561 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     7621 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatgacc gctttggaaa
     7681 aacaaagact gtatttcctg gaaattaatg tttattcaat aaactgtg
//
