LOCUS       sequence-115894-        5999 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RMCE donor plasmid for FlpO. the marker gene is green fluorescent 
            (mWasabi) with PA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5999)
  AUTHORS   Callesen MM, Berthelsen MF, Lund S, Fuchtbauer AC, Fuchtbauer EM, 
            Jakobsen JE
  TITLE     Recombinase-Mediated Cassette Exchange (RMCE)-in Reporter Cell Lines
            as an Alternative to the Flp-in System.
  JOURNAL   PLoS One. 2016 Aug 19;11(8):e0161471. doi: 
            10.1371/journal.pone.0161471. eCollection 2016.
  PUBMED    27541869
REFERENCE   2  (bases 1 to 5999)
  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: "PLoS One.";
            date: "2016-08-19"; volume: "11(8):e0161471. doi"; pages: " 
            10.1371/journal.pone.0161471. eCollection 2016"
COMMENT     Sequence Label: Frt-Atg-Hygro-CMV-green (mWasabi)-PA-F3
FEATURES             Location/Qualifiers
     source          1..5999
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     protein_bind    187..234
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             242..1264
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             242..1264
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    1394..1515
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        1564..1943
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1944..2147
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2098..2118
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2192..2211
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2192..2210
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2320..2329
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2326..3030
                     /codon_start=1
                     /product="green variant of teal fluorescent protein mTFP1 
                     (Ai et al., 2008)"
                     /label=mWasabi
                     /translation="MVSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYDG
                     TNTINLEVKEGAPLPFSYDILTTAFSYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMTF
                     EDKGIVKVKSDISMEEDSFIYEIHLKGENFPPNGPVMQKETTGWDASTERMYVRDGVLK
                     GDVKMKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYEIA
                     VARNSTDGMDELY"
     protein_bind    complement(3135..3168)
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     primer_bind     complement(3214..3231)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3220..3444
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3577..3596)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3672..3793
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3842..3858)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3842..3858)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3855..3877)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3866..3882
                     /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(3890..3896,3897..3914,3915..3920))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3935..3956
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4073..4090)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4244..4832)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4324..4343)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5003..5794,5795..5863))
                     /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     5626..5645
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5864..5968)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgggtg atggttcacg
      181 tacctagaag ttcctattcc gaagttccta ttctctagaa agtataggaa cttccttggc
      241 caaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag
      301 cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt
      361 aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg
      421 ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg
      481 ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca
      541 agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc
      601 gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat
      661 cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca
      721 ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct
      781 gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc
      841 caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat
      901 gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg
      961 tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg
     1021 gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg
     1081 caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc
     1141 cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg
     1201 tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga
     1261 atagcacgta ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     1321 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     1381 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     1441 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     1501 catcaatgta tcttatcatg tctgtatacc gtcgtcgcga tgtacgggcc agatatacgc
     1561 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
     1621 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     1681 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     1741 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     1801 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     1861 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     1921 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
     1981 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
     2041 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
     2101 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta
     2161 gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag
     2221 ctggctagcg tttaaactta agcttggtac cgagctcgga tccactagtc cagtgtggtg
     2281 gaattctgca gatatccagc acagtggcgg ccgctcgagg ccaccatggt gagcaagggc
     2341 gaggagacca caatgggcgt aatcaagccc gacatgaaga tcaagctgaa gatggagggc
     2401 aacgtgaatg gccacgcctt cgtgatcgag ggcgagggcg agggcaagcc ctacgacggc
     2461 accaacacca tcaacctgga ggtgaaggag ggagcccccc tgcccttctc ctacgacatt
     2521 ctgaccaccg cgttcagtta cggcaacagg gccttcacca agtaccccga cgacatcccc
     2581 aactacttca agcagtcctt ccccgagggc tactcttggg agcgcaccat gaccttcgag
     2641 gacaagggca tcgtgaaggt gaagtccgac atctccatgg aggaggactc cttcatctac
     2701 gagatacacc tcaagggcga gaacttcccc cccaacggcc ccgtgatgca gaaggagacc
     2761 accggctggg acgcctccac cgagaggatg tacgtgcgcg acggcgtgct gaagggcgac
     2821 gtcaagatga agctgctgct ggagggcggc ggccaccacc gcgttgactt caagaccatc
     2881 tacagggcca agaaggcggt gaagctgccc gactatcact ttgtggacca ccgcatcgag
     2941 atcctgaacc acgacaagga ctacaacaag gtgaccgttt acgagatcgc cgtggcccgc
     3001 aactccaccg acggcatgga cgagctgtac cgagctcgga tccactagtc cagtgtggtg
     3061 gaattctgca gatatccagc acagtggcgg ccgctcgagt ctagagggcc cgtttgcttt
     3121 aattaacctg cagggaagtt cctatactat ttgaagaata ggaacttcgg aataggaact
     3181 tcacatgtgg gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc
     3241 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     3301 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     3361 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     3421 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg
     3481 ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     3541 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     3601 ttctcgccac gttcgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     3661 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     3721 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     3781 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     3841 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     3901 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     3961 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     4021 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     4081 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     4141 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     4201 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4261 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     4321 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4381 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     4441 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     4501 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4561 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     4621 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     4681 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     4741 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     4801 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     4861 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     4921 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     4981 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     5041 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     5101 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     5161 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     5221 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     5281 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     5341 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     5401 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     5461 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     5521 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     5581 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     5641 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     5701 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     5761 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     5821 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     5881 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     5941 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
