LOCUS       sequence-173521-        6102 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FlpO under pCAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6102)
  AUTHORS   Weinberg BH, Pham NT, Caraballo LD, Lozanoski T, Engel A, Bhatia S, 
            Wong WW
  TITLE     Large-scale design of robust genetic circuits with multiple inputs 
            and outputs for mammalian cells.
  JOURNAL   Nat Biotechnol. 2017 Mar 27. doi: 10.1038/nbt.3805.
  PUBMED    28346402
REFERENCE   2  (bases 1 to 6102)
  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 
            Biotechnol. 2017 Mar 27. doi: 10.1038/nbt.3805."
COMMENT     Sequence Label: pCAG-FlpO
FEATURES             Location/Qualifiers
     source          1..6102
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(1..792,793..861))
                     /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     624..643
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(862..966)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        997..1376
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1378..1653
                     /label=chicken beta-actin promoter
     intron          1654..2669
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2593..2615
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2677..2696
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2747..2756
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2753..2758,2759..2779,2780..4051)
                     /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(4137..4156)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4202..4257
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4256..4275)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4618..4634)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4618..4634)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4631..4653)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4642..4658
                     /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(4666..4672,4673..4690,4691..4696))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4711..4732
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      4817..4951
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4971..5105
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5008..5027)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5062..5081
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5173..5190)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5344..5932)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5424..5443)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
       61 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
      121 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
      181 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
      241 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
      301 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
      361 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
      421 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
      481 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
      541 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
      601 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
      661 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
      721 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
      781 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
      841 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
      901 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
      961 tccgcgcaca tttccccgaa aagtgccacc tgggtcgaca ttgattattg actagttatt
     1021 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
     1081 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
     1141 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     1201 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
     1261 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
     1321 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg
     1381 aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt
     1441 tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggcgc
     1501 gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggaa aggtgcggcg
     1561 gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg
     1621 cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgttgcc ttcgccccgt
     1681 gccccgctcc gcgccgcctc gcgccgcccg ccccggctct gactgaccgc gttactccca
     1741 caggtgagcg ggcgggacgg cccttctcct ccgggctgta attagcgctt ggtttaatga
     1801 cggctcgttt cttttctgtg gctgcgtgaa agccttaaag ggctccggga gggccctttg
     1861 tgcggggggg agcggctcgg ggggtgcgtg cgtgtgtgtg tgcgtgggga gcgccgcgtg
     1921 cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc tttgtgcgct
     1981 ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc ccgcggtgcg gggggggctg
     2041 cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag ggggtgtggg
     2101 cgcggcggtc gggctgtaac ccccccctgc acccccctcc ccgagttgct gagcacggcc
     2161 cggcttcggg tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg
     2221 ggtggcggca ggtgggggtg ccgggcgggg cggggccgcc tcgggccggg gagggctcgg
     2281 gggaggggcg cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc
     2341 attgcctttt atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctgtg
     2401 cggagccgaa atctgggagg cgccgccgca ccccctctag cgggcgcggg gcgaagcggt
     2461 gcggcgccgg caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc
     2521 cccttctccc tctccagcct cggggctgtc cgcgggggga cggctgcctt cgggggggac
     2581 ggggcagggc ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc
     2641 atgttcatgc cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt
     2701 ctcatcattt tggcaaagaa ttgattaatt cgagcgaacg cgtgccgcca ccatggctcc
     2761 taagaagaag aggaaggtga tgagccagtt cgacatcctg tgcaagaccc cccccaaggt
     2821 gctggtgcgg cagttcgtgg agagattcga gaggcccagc ggcgagaaga tcgccagctg
     2881 tgccgccgag ctgacctacc tgtgctggat gatcacccac aacggcaccg ccatcaagag
     2941 ggccaccttc atgagctaca acaccatcat cagcaacagc ctgagcttcg acatcgtgaa
     3001 caagagcctg cagttcaagt acaagaccca gaaggccacc atcctggagg ccagcctgaa
     3061 gaagctgatc cccgcctggg agttcaccat catcccttac aacggccaga agcaccagag
     3121 cgacatcacc gacatcgtgt ccagcctgca gctgcagttc gagagcagcg aggaggccga
     3181 caagggcaac agccacagca agaagatgct gaaggccctg ctgtccgagg gcgagagcat
     3241 ctgggagatc accgagaaga tcctgaacag cttcgagtac accagcaggt tcaccaagac
     3301 caagaccctg taccagttcc tgttcctggc cacattcatc aactgcggca ggttcagcga
     3361 catcaagaac gtggacccca agagcttcaa gctggtgcag aacaagtacc tgggcgtgat
     3421 cattcagtgc ctggtgaccg agaccaagac aagcgtgtcc aggcacatct actttttcag
     3481 cgccagaggc aggatcgacc ccctggtgta cctggacgag ttcctgagga acagcgagcc
     3541 cgtgctgaag agagtgaaca ggaccggcaa cagcagcagc aacaagcagg agtaccagct
     3601 gctgaaggac aacctggtgc gcagctacaa caaggccctg aagaagaacg ccccctaccc
     3661 catcttcgct atcaagaacg gccctaagag ccacatcggc aggcacctga tgaccagctt
     3721 tctgagcatg aagggcctga ccgagctgac aaacgtggtg ggcaactgga gcgacaagag
     3781 ggcctccgcc gtggccagga ccacctacac ccaccagatc accgccatcc ccgaccacta
     3841 cttcgccctg gtgtccaggt actacgccta cgaccccatc agcaaggaga tgatcgccct
     3901 gaaggacgag accaacccca tcgaggagtg gcagcacatc gagcagctga agggcagcgc
     3961 cgagggcagc atcagatacc ccgcctggaa cggcatcatc agccaggagg tgctggacta
     4021 cctgagcagc tacatcaaca ggcggatctg agcggccgca ctcctcaggt gcaggctgcc
     4081 tatcagaagg tggtggctgg tgtggccaat gccctggctc acaaatacca ctgagatctt
     4141 tttccctctg ccaaaaatta tggggacatc atgaagcccc ttgagcatct gacttctggc
     4201 taataaagga aatttatttt cattgcaata gtgtgttgga attttttgtg tctctcactc
     4261 ggaaggacat atgggagggc aaatcattta aaacatcaga atgagtattt ggtttagagt
     4321 ttggcaacat atgcccatat gctggctgcc atgaacaaag gttggctata aagaggtcat
     4381 cagtatatga aacagccccc tgctgtccat tccttattcc atagaaaagc cttgacttga
     4441 ggttagattt tttttatatt ttgttttgtg ttattttttt ctttaacatc cctaaaattt
     4501 tccttacatg ttttactagc cagatttttc ctcctctcct gactactccc agtcatagct
     4561 gtccctcttc tcttatggag atccctcgac ctgcagccca agcttggcgt aatcatggtc
     4621 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     4681 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     4741 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagcggatcc gcatctcaat
     4801 tagtcagcaa ccatagtccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     4861 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     4921 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct aacttgttta
     4981 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     5041 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     5101 ggatccgctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     5161 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     5221 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     5281 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     5341 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     5401 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     5461 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     5521 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     5581 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     5641 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     5701 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     5761 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     5821 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5881 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5941 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     6001 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     6061 taaatcaatc taaagtatat atgagtaaac ttggtctgac ag
//
