LOCUS       sequence-393482-        5976 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  hygR selection nls-mScarlet marked rapid RMCE integration vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5976)
  AUTHORS   Nonet ML
  TITLE     Rapid generation of C. elegans single-copy transgenes combining RMCE
            and drug selection.
  JOURNAL   Genetics. 2023 Apr 20:iyad072. doi: 10.1093/genetics/iyad072.
  PUBMED    37079426
REFERENCE   2  (bases 1 to 5976)
  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. 
            2023 Apr 20:iyad072. doi: 10.1093/genetics/iyad072."
COMMENT     Sequence Label: pHygR2r
FEATURES             Location/Qualifiers
     source          1..5976
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(100..117)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(271..859)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(351..370)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1030..1821,1822..1890))
                     /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     1653..1672
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1891..1995)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2021..2476)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2158..2179)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2370..2389
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    complement(2488..2521)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(2601..2634)
                     /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    2655..2688
                     /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)"
     CDS             2701..2721
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    4520..4544
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(4545..5570)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(4545..5570)
                     /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(5573..5597)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 atcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
       61 cggggagagg cggtttgcgt attgggcgaa ttccgcttcc tcgctcactg actcgctgcg
      121 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
      181 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
      241 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
      301 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
      361 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
      421 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
      481 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccat
      541 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
      601 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
      661 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
      721 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
      781 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
      841 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
      901 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
      961 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     1021 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     1081 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     1141 atctggcccc agtgctgcaa tgataccgcg agatccacgc tcaccggctc cagatttatc
     1201 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     1261 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     1321 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     1381 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     1441 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     1501 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     1561 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     1621 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     1681 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     1741 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     1801 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     1861 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     1921 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     1981 aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt
     2041 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     2101 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     2161 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     2221 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     2281 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     2341 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     2401 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     2461 ccgctacagg gcgcgtcgtc ggagctcata acttcgtata gcatacatta tacgaagtta
     2521 tgcggccgct ctagaactag tggtaccgaa gagcatgcaa cgcgtggtta cctgcaggct
     2581 cttcgccact taagacccgg gaagttccta tactatttga agaataggaa cttctgcagg
     2641 aattcgatat cggtgaagtt cctattctct agaaagtata ggaacttcaa aatgggatcc
     2701 ccaaagaaga agcgtaaggt gtcgatggtt tccaagggag aggccgtcat caaggagttc
     2761 atgcgtttca aggtccacat ggagggatcc atgaacggac acgagttcga gatcgaggga
     2821 gagggagagg gacgtccata cgagggaacc caaaccgcca agctcaaggt caccaaggta
     2881 agtttaaaca tatatatact aactaaccct gattatttaa attttcaggg aggaccactc
     2941 ccattctcct gggacatcct ctccccacaa ttcatgtacg gatcccgtgc cttcaccaag
     3001 cacccagccg acatcccaga ctactacaag caatccttcc cagagggatt caagtgggag
     3061 cgtgtcatga acttcgagga cggaggagcc gtcaccgtca cccaagacac ctccctcgag
     3121 gacggaaccc tcatctacaa ggtaagttta aacagttcgg tactaactaa ccatacatat
     3181 ttaaattttc aggtcaagct ccgtggaacc aacttcccac cagacggacc agtcatgcaa
     3241 aagaagacca tgggatggga ggcctccacc gagcgtctct acccagagga cggagtcctc
     3301 aagggagaca tcaagatggc cctccgtctc aaggacggag gacgttacct cgccgacttc
     3361 aaggtaagtt taaacatgat tttactaact aactaatctg atttaaattt tcagaccacc
     3421 tacaaggcca agaagccagt ccaaatgcca ggagcctaca acgtcgaccg taagctcgac
     3481 atcacctccc acaacgagga ctacaccgtc gtcgagcaat acgagcgttc cgagggacgt
     3541 cactccaccg gaggaatgga cgagctctac aaggcctccc gtcgtcgtaa ggccaaccca
     3601 accaagctct ccgagaacgc caagaagctc gccaagtaga tcaactgctc taattccatc
     3661 tcgtttctct cttttctttc aatctcttta cccgtttctc ttcccctacg tcccccaaac
     3721 ctaccactgc acagtaacac atacacattc aacgtgtcac aataaaagcc catcaacacc
     3781 ttctctgttg ttttttttct tcaaatgcat ttttttcact ttgtttaata atttttacgc
     3841 atcggatttg taggatagtg gaaatttgtc cttcaatgct tcaattgatt cgacacttct
     3901 caatattttt gatcaataaa ccattgattt tctatttaga agaagatcac tgtaaataac
     3961 accaaaaaca tcacagcaaa aaaactccca ctcaattata ttactgtttt tattgaaact
     4021 agaataaatt tgaaaatctt caaagcatac acaattaaaa aaaacgaaaa aagaaaaagt
     4081 cccgttgatt ctcaaactta aatttttaaa cttgtctgaa ccatgttgta agtttgtaaa
     4141 agtttcccag ttgtcctcga acaggtctaa aaattgcggt cataaactga aacgtaacat
     4201 atgataaggt attttgtgtg cggttttttc tatgatgttt caaaaaaatt tatcagaagt
     4261 aaaaaacata agaagtgtac acagaacatt gtgtagattt tgggatagaa ttattattaa
     4321 tggggggagg gagcacaatt tttttcgact ttttattacg aattaattct atttttgaat
     4381 ctacacaatt tcattgttag aggtgactta aaagaagcta aaaaacaaag aaattaagag
     4441 aagttttttg ataataacaa aaataggggg tgggagcaca gggagaaaga gcatgtaggg
     4501 atgttgaaga gtaattggac ccactttgta caagaaagct gggtctattc ctttgccctc
     4561 ggacgagtgc tggggcgtcg gtttccacta tcggcgagta cttctacaca gccatcggtc
     4621 cagacggccg cgcttctgcg ggcgatttgt gtacgcccga cagtcccggc tccggatcgg
     4681 acgattgcgt cgcatcgacc ctgcgcccaa gctgcatcat cgaaattgcc gtcaaccaag
     4741 ctctgataga gttggtcaag accaatgcgg agcatatacg cccggagccg cggcgatcct
     4801 gcaagctccg gatgcctccg ctcgaagtag cgcgtctgct gctccataca agccaaccac
     4861 ggcctccaga agaagatgtt ggcgacctcg tattgggaat ccccgaacat cgcctcgctc
     4921 cagtcaatga ccgctgttat gcggccattg tccgtcagga cattgttgga gccgaaatcc
     4981 gcgtgcacga ggtgccggac ttcggggcag tcctcggccc aaagcatcag ctcatcgaga
     5041 gcctgcgcga cggacgcact gacggtgtcg tccatcacag tttgccagtg atacacatgg
     5101 ggatcagcaa tcgcgcatat gaaatcacgc catgtagtgt attgaccgat tccttgcggt
     5161 ccgaatgggc cgaacccgct cgtctggcta agatcggccg cagcgatcgc atccatggcc
     5221 tccgcgaccg gctgcagaac agcgggcagt tcggtttcag gcaggtcttg caacgtgaca
     5281 ccctgtgcac ggcgggagat gcaataggtc aggctctcgc tgaattcccc aatgtcaagc
     5341 acttccggaa tcgggagcgc ggccgatgca aagtgccgat aaacataacg atctttgtag
     5401 aaaccatcgg cgcagctatt tacccgcagg acatatccac gccctcctac atcgaagctg
     5461 aaagcacgag attcttcgcc ctccgagagc tgcatcaggt cggagacgct gtcgaacttt
     5521 tcgatcagaa acttctcgac agacgtcgcg gtgagttcag gctttttcat ggagcctgct
     5581 tttttgtaca aacttgcggc accgcctgac atattacctt aaaattcaaa aattaatttc
     5641 agatcagtca taaccaagaa aacaaaataa acgttaggac agaaaattca aattaattaa
     5701 aaattatgct gaataagaaa attacatgca aagccttatt ttcctcatag aaacagttga
     5761 ataacatatt ttttaattga aaacatagca aaattcgtgg taaaatttgt caaaaccttg
     5821 gaaaaaatcg aaaaaagaac atttcccgat gcattttcct atttctcgtt gccgacaacg
     5881 tataaacgtt gaatactcag tcaacgaatt tggctgctgg cttggactct cgcacggaag
     5941 agacgcgtgt gtagatttac gacgaaagca aaaatg
//
