LOCUS       sequence-393496-        5942 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  hygR selection nls-mScarlet marked rapid RMCE integration vector 
            without loxP site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5942)
  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 5942)
  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: pHygR8
FEATURES             Location/Qualifiers
     source          1..5942
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    887..911
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(912..1937)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(912..1937)
                     /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(1940..1964)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2443..2460)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2614..3202)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2694..2713)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3373..4164,4165..4233))
                     /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     3996..4015
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4234..4338)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4364..4819)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4501..4522)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4713..4732
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    complement(4910..4943)
                     /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    4964..4997
                     /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             5010..5030
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 aagtagatca actgctctaa ttccatctcg tttctctctt ttctttcaat ctctttaccc
       61 gtttctcttc ccctacgtcc cccaaaccta ccactgcaca gtaacacata cacattcaac
      121 gtgtcacaat aaaagcccat caacaccttc tctgttgttt tttttcttca aatgcatttt
      181 tttcactttg tttaataatt tttacgcatc ggatttgtag gatagtggaa atttgtcctt
      241 caatgcttca attgattcga cacttctcaa tatttttgat caataaacca ttgattttct
      301 atttagaaga agatcactgt aaataacacc aaaaacatca cagcaaaaaa actcccactc
      361 aattatatta ctgtttttat tgaaactaga ataaatttga aaatcttcaa agcatacaca
      421 attaaaaaaa acgaaaaaag aaaaagtccc gttgattctc aaacttaaat ttttaaactt
      481 gtctgaacca tgttgtaagt ttgtaaaagt ttcccagttg tcctcgaaca ggtctaaaaa
      541 ttgcggtcat aaactgaaac gtaacatatg ataaggtatt ttgtgtgcgg ttttttctat
      601 gatgtttcaa aaaaatttat cagaagtaaa aaacataaga agtgtacaca gaacattgtg
      661 tagattttgg gatagaatta ttattaatgg ggggagggag cacaattttt ttcgactttt
      721 tattacgaat taattctatt tttgaatcta cacaatttca ttgttagagg tgacttaaaa
      781 gaagctaaaa aacaaagaaa ttaagagaag ttttttgata ataacaaaaa tagggggtgg
      841 gagcacaggg agaaagagca tgtagggatg ttgaagagta attggaccca ctttgtacaa
      901 gaaagctggg tctattcctt tgccctcgga cgagtgctgg ggcgtcggtt tccactatcg
      961 gcgagtactt ctacacagcc atcggtccag acggccgcgc ttctgcgggc gatttgtgta
     1021 cgcccgacag tcccggctcc ggatcggacg attgcgtcgc atcgaccctg cgcccaagct
     1081 gcatcatcga aattgccgtc aaccaagctc tgatagagtt ggtcaagacc aatgcggagc
     1141 atatacgccc ggagccgcgg cgatcctgca agctccggat gcctccgctc gaagtagcgc
     1201 gtctgctgct ccatacaagc caaccacggc ctccagaaga agatgttggc gacctcgtat
     1261 tgggaatccc cgaacatcgc ctcgctccag tcaatgaccg ctgttatgcg gccattgtcc
     1321 gtcaggacat tgttggagcc gaaatccgcg tgcacgaggt gccggacttc ggggcagtcc
     1381 tcggcccaaa gcatcagctc atcgagagcc tgcgcgacgg acgcactgac ggtgtcgtcc
     1441 atcacagttt gccagtgata cacatgggga tcagcaatcg cgcatatgaa atcacgccat
     1501 gtagtgtatt gaccgattcc ttgcggtccg aatgggccga acccgctcgt ctggctaaga
     1561 tcggccgcag cgatcgcatc catggcctcc gcgaccggct gcagaacagc gggcagttcg
     1621 gtttcaggca ggtcttgcaa cgtgacaccc tgtgcacggc gggagatgca ataggtcagg
     1681 ctctcgctga attccccaat gtcaagcact tccggaatcg ggagcgcggc cgatgcaaag
     1741 tgccgataaa cataacgatc tttgtagaaa ccatcggcgc agctatttac ccgcaggaca
     1801 tatccacgcc ctcctacatc gaagctgaaa gcacgagatt cttcgccctc cgagagctgc
     1861 atcaggtcgg agacgctgtc gaacttttcg atcagaaact tctcgacaga cgtcgcggtg
     1921 agttcaggct ttttcatgga gcctgctttt ttgtacaaac ttgcggcacc gcctgacata
     1981 ttaccttaaa attcaaaaat taatttcaga tcagtcataa ccaagaaaac aaaataaacg
     2041 ttaggacaga aaattcaaat taattaaaaa ttatgctgaa taagaaaatt acatgcaaag
     2101 ccttattttc ctcatagaaa cagttgaata acatattttt taattgaaaa catagcaaaa
     2161 ttcgtggtaa aatttgtcaa aaccttggaa aaaatcgaaa aaagaacatt tcccgatgca
     2221 ttttcctatt tctcgttgcc gacaacgtat aaacgttgaa tactcagtca acgaatttgg
     2281 ctgctggctt ggactctcgc acggaagaga cgcgtgtgta gatttacgac gaaagcaaaa
     2341 atgatcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     2401 gcgcggggag aggcggtttg cgtattgggc gaattccgct tcctcgctca ctgactcgct
     2461 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     2521 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     2581 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     2641 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     2701 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     2761 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     2821 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     2881 cattcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     2941 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     3001 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     3061 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     3121 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     3181 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     3241 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     3301 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     3361 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     3421 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     3481 accatctggc cccagtgctg caatgatacc gcgagatcca cgctcaccgg ctccagattt
     3541 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     3601 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     3661 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     3721 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     3781 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     3841 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     3901 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     3961 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     4021 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     4081 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     4141 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     4201 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     4261 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     4321 acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat
     4381 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     4441 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     4501 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     4561 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     4621 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     4681 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     4741 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     4801 gcgccgctac agggcgcgtc gtcggagctc caccgcggtg gcggccgctc tagaactagt
     4861 ggcgaagagc ctgcaggtaa ccacgcgttg catgctcttc ggtacccggg aagttcctat
     4921 actatttgaa gaataggaac ttctgcagga attcgatatc ggtgaagttc ctattctcta
     4981 gaaagtatag gaacttcaaa atgggatccc caaagaagaa gcgtaaggtg tcgatggttt
     5041 ccaagggaga ggccgtcatc aaggagttca tgcgtttcaa ggtccacatg gagggatcca
     5101 tgaacggaca cgagttcgag atcgagggag agggagaggg acgtccatac gagggaaccc
     5161 aaaccgccaa gctcaaggtc accaaggtaa gtttaaacat atatatacta actaaccctg
     5221 attatttaaa ttttcaggga ggaccactcc cattctcctg ggacatcctc tccccacaat
     5281 tcatgtacgg atcccgtgcc ttcaccaagc acccagccga catcccagac tactacaagc
     5341 aatccttccc agagggattc aagtgggagc gtgtcatgaa cttcgaggac ggaggagccg
     5401 tcaccgtcac ccaagacacc tccctcgagg acggaaccct catctacaag gtaagtttaa
     5461 acagttcggt actaactaac catacatatt taaattttca ggtcaagctc cgtggaacca
     5521 acttcccacc agacggacca gtcatgcaaa agaagaccat gggatgggag gcctccaccg
     5581 agcgtctcta cccagaggac ggagtcctca agggagacat caagatggcc ctccgtctca
     5641 aggacggagg acgttacctc gccgacttca aggtaagttt aaacatgatt ttactaacta
     5701 actaatctga tttaaatttt cagaccacct acaaggccaa gaagccagtc caaatgccag
     5761 gagcctacaa cgtcgaccgt aagctcgaca tcacctccca caacgaggac tacaccgtcg
     5821 tcgagcaata cgagcgttcc gagggacgtc actccaccgg aggaatggac gagctctaca
     5881 aggcctcccg tcgtcgtaag gccaacccaa ccaagctctc cgagaacgcc aagaagctcg
     5941 cc
//
