LOCUS       sequence-306102-        7855 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RMCE integration vector with self excising cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7855)
  AUTHORS   Nonet ML
  TITLE     Efficient Transgenesis in Caenorhabditis elegans Using Flp 
            Recombinase-Mediated Cassette Exchange.
  JOURNAL   Genetics. 2020 Jun 8. pii: genetics.120.303388. doi: 
            10.1534/genetics.120.303388.
  PUBMED    32513816
REFERENCE   2  (bases 1 to 7855)
  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: "Genetics. 
            2020 Jun 8. pii: genetics.120.303388. doi: 
            10.1534/genetics.120.303388."
COMMENT     Sequence Label: pLF3FShC
FEATURES             Location/Qualifiers
     source          1..7855
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     78..100
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     92..109
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     93..109
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     119..138
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        119..137
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      1731..2054
                     /gene="tbb-2"
                     /label=tbb-2 terminator
                     /note="transcription terminator for the C. elegans tubulin 
                     beta-2 gene"
     CDS             complement(3871..4896)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(3871..4896)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     protein_bind    complement(4897..4930)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    4951..4984
                     /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)"
     protein_bind    5082..5115
                     /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(5161..5181)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5161..5179)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5200..5216)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5200..5216)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5213..5235)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5224..5240
                     /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(5248..5254,5255..5272,5273..5278))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5293..5314
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5430..5447)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5601..6189)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5681..5700)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6360..7151,7152..7220))
                     /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     6983..7002
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7221..7325)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7351..7806)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7488..7509)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7700..7719
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg
       61 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg agcgcgcgta
      121 atacgactca ctatagggcg aattgggtac cgggcccccg gttaacgggt cgacggtatc
      181 gataagctta cgatcaccaa aaacggaacg ttgagctgga cggaaatagt ggtaaagtga
      241 catgattata gtttgaagat ttctaatttc acaattagag caaatgttgt tcggtattta
      301 ttttcaacgg tatttatact attttccacc tttttctaga acattcgagc tgcttgttgc
      361 aaaaggaggg cgactcacat tcggtacatg gaaaagtagt gtacacaata aagagaccca
      421 gatacatttt ccgtctgcgt ctctttgcac ccaccgggag tattttcaaa cgaatgcatc
      481 taggaccttc tagaacattc tgtaaggctg cagaatgcgg gtatataagg aaagcgggct
      541 cagaggaagc caacacgctt tgttctagtg catctaaaaa acttcgaaaa tatgtcaaat
      601 cttctcactg ttcatcaaaa tcttccagca cttccagttg acgcgacaag tgatgaagtt
      661 cgcaagaacc tcatggatat gttccgtgac agacaagcct tctccgagca cacctggaag
      721 atgcttctct cagtgtgccg ttcttgggct gcctggtgca agcttaacaa ccgcaagtgg
      781 ttcccagccg agccagaaga cgttcgtgac tacttgctct acctccaagc tcgtggattg
      841 gcggtgaaga cgatccaaca acatctcgga cagctcaata tgctccatcg ccgatctgga
      901 cttccacgcc catcagattc taatgccgta tctctcgtaa tgagacgtat ccgcaaggaa
      961 aatgttgatg ctggagagag agccaagcaa gccctcgcct tcgaaaggta agtttaaata
     1021 ttaatgtatt caatcctaag acttgacgaa attttcagaa ccgacttcga tcaggtccgt
     1081 tctcttatgg aaaattccga tcgttgccag gacatccgaa acctcgcctt cctcggaatt
     1141 gcttacaaca cacttctccg tatcgccgaa attgcccgca ttcgcgtcaa ggatattagt
     1201 cgcacagacg gaggaagaat gctcatccac atcggacgta caaaaacgct ggtctcgact
     1261 gccggagtcg aaaaagcgct ctcccttgga gttaccaagc tcgttgagcg ctggatttcg
     1321 gtttccggag tcgccgatga tccaaacaac tacctgttct gtcgtgttcg caagaacgga
     1381 gttgctgccc cgagtgcaac atcacaactc tcgactcgag ctcttgaagg taagtttaaa
     1441 caacatatta gctaagaata agctgaaaat aaattttcag gaatcttcga agctacccac
     1501 cgtctcatct acggagccaa ggatgattcc ggacaacgtt acttggcttg gtctggacat
     1561 tccgcccgtg tcggagctgc ccgtgacatg gctcgcgctg gagtttccat cccagagatt
     1621 atgcaagccg gaggttggac caacgtcaac attgtgatga actatattag aaacttggat
     1681 tctgaaactg gcgccatggt tcgtcttctc gaagacggtg attaagataa atgcaaaatc
     1741 ctttcaagca ttcccttctt ctctatcact cttctttctt tttgtcaaaa aattctctcg
     1801 ctaatttatt tgctttttta atgttattat tttatgactt tttatagtca ctgaaaagtt
     1861 tgcatctgag tgaagtgaat gctatcaaaa tgtgattctg tctgatgtac tttcacaatc
     1921 tctcttcaat tccattttga agtgctttaa acccgaaagg ttgagaaaaa tgcgagcgct
     1981 caaatatttg tattgtgttc gttgagtgac ccaacaaaaa gaggaaactt tattgtgccg
     2041 ccaagaaaaa agtctcactg aatctcaaat attttattag aaaacaccaa caatttagat
     2101 atttctgtat ccacggttgg cgttgacagc ctctgatggt ggagacttgt ctgggcactg
     2161 gcagtactcg gcatcctttc ctggtcttcc gtctggtcca gtctctccct gggttccgtc
     2221 tggtccttga cgtccttgct ttccagatgg tccttctggt ccaacggttc ctggctctcc
     2281 tggtggtccg tgaagaccgt tctgtccagc tggtcctgtt ggtccttgat ctcctcttgg
     2341 tccctttggt cctggtcttc cgattggatg ctcggcgtcg cgtcccttct ctcctggtgg
     2401 tcctggctct ccgatctttc cgatttgacc ggatgatcct tgctctcctg gatgtcctgg
     2461 ttgtccatct cttcctgggt ttccattttg tcctcttggt cctggaagtc cacgtggtcc
     2521 tggtcttcca agagctcctg gtggtccaac tggtccttgt gggcaagtga agcatccgac
     2581 agactcgcgt tgtggagcga tatcatcagc tccaactcct ggaacaccgt caagtcctgg
     2641 aactccgtcg agtcctggaa ctcctggaac tccctttggt ccggatggtc cagctgggca
     2701 cttgttatca gcattgcagt tgcaggttgg tccatttcca ttggctccat ttggagtctt
     2761 tggctggttg aagactggtg gaactgctgg tggttgacct ggtggagcag atggggatcc
     2821 atcagcatgt ggagttgggg tagcgttggt ctcttcatat tgacaacgga cacgcttgct
     2881 agatcttccg atgacaacta tgtccttcca caatccattg gtagattgct agaaaattaa
     2941 aaattattta aaattcaaaa tataatttaa ctcactctga acacattcat ctcggtatca
     3001 agcttctctc tgaatccatc aacctccgag tagatggttg acatggtcat gaagcatacc
     3061 ataagagtgg cgacagtgac cgaagccgtc acataacacg caagttttac agacatctct
     3121 gaaattatca aatttaggat ttcaaaataa ttcggccaaa gaaatgtaac tcacctagtc
     3181 ttcgagaagc agagacttgt cgatttccta ccggattcgt ctccttttat taggaaatct
     3241 ttccgcaagt tttcaagttt tttttgtttt ttttttacat taccgacgag acagtgtgcg
     3301 agacgcagag acgataaaag actggataag aacgggagat aaggagtacg gatgtaggtg
     3361 aggaatgtgg ccgaaaggac agatgggctt tattcgggaa gattggttgg gaaatgaacc
     3421 agagaagaat gactgaataa agttgttgaa acttggtgag aatggaagat agacaatcga
     3481 gggcagctag tttccttcct cctctatata tttttatgtt ttcttttaaa ccttcctcca
     3541 tatttctgaa ttgaatgttg ttgcatattg tgtgtcctta tttcatttcc atcatccttt
     3601 tgtgtgtttc ccccttccac caattccccc cacgtcctaa tgaaatgaaa gcgagaagat
     3661 aaataaactt gcacattgtg ttcagatgag aggagcggaa tacgaattgg gagacggaaa
     3721 gagaaaaaaa aatgtttaga tttggattga attaattttt tagaaagcaa gaaaaaaaac
     3781 acaaagagaa taaataaata ataggaaaaa aaacaaatat tcagtattta aaatttgaaa
     3841 attcaacgac gttggcgtcg atcatcccgc ctattccttt gccctcggac gagtgctggg
     3901 gcgtcggttt ccactatcgg cgagtacttc tacacagcca tcggtccaga cggccgcgct
     3961 tctgcgggcg atttgtgtac gcccgacagt cccggctccg gatcggacga ttgcgtcgca
     4021 tcgaccctgc gcccaagctg catcatcgaa attgccgtca accaagctct gatagagttg
     4081 gtcaagacca atgcggagca tatacgcccg gagccgcggc gatcctgcaa gctccggatg
     4141 cctccgctcg aagtagcgcg tctgctgctc catacaagcc aaccacggcc tccagaagaa
     4201 gatgttggcg acctcgtatt gggaatcccc gaacatcgcc tcgctccagt caatgaccgc
     4261 tgttatgcgg ccattgtccg tcaggacatt gttggagccg aaatccgcgt gcacgaggtg
     4321 ccggacttcg gggcagtcct cggcccaaag catcagctca tcgagagcct gcgcgacgga
     4381 cgcactgacg gtgtcgtcca tcacagtttg ccagtgatac acatggggat cagcaatcgc
     4441 gcatatgaaa tcacgccatg tagtgtattg accgattcct tgcggtccga atgggccgaa
     4501 cccgctcgtc tggctaagat cggccgcagc gatcgcatcc atggcctccg cgaccggctg
     4561 cagaacagcg ggcagttcgg tttcaggcag gtcttgcaac gtgacaccct gtgcacggcg
     4621 ggagatgcaa taggtcaggc tctcgctgaa ttccccaatg tcaagcactt ccggaatcgg
     4681 gagcgcggcc gatgcaaagt gccgataaac ataacgatct ttgtagaaac catcggcgca
     4741 gctatttacc cgcaggacat atccacgccc tcctacatcg aagctgaaag cacgagattc
     4801 ttcgccctcc gagagctgca tcaggtcgga gacgctgtcg aacttttcga tcagaaactt
     4861 ctcgacagac gtcgcggtga gttcaggctt tttcatgaag ttcctatact ttctagagaa
     4921 taggaacttc accgatatcg aattcctgca gaagttccta ttcttcaaat agtataggaa
     4981 cttcccgggt accgaagagc atgcaacgcg tggttacctg caggctcttc gccactagtt
     5041 ctagagcggc cgccaccgcg gtggagctcc aattcgcccg tataacttcg tataatgtat
     5101 gctatacgaa gttatccatc tagagcggcc gccaccgcgg tggagctcca gcttttgttc
     5161 cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     5221 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     5281 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     5341 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     5401 cggtttgcgt attgggcgaa ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     5461 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     5521 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     5581 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     5641 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5701 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5761 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5821 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccat tcagcccgac
     5881 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5941 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     6001 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     6061 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     6121 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     6181 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     6241 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     6301 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     6361 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     6421 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     6481 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     6541 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     6601 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     6661 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     6721 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     6781 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     6841 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     6901 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     6961 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     7021 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     7081 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     7141 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     7201 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     7261 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     7321 ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat
     7381 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     7441 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     7501 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     7561 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     7621 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     7681 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     7741 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     7801 gcgcgtccca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcg
//
