LOCUS       sequence-393498-        5966 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 5966)
  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 5966)
  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: pHygR2
FEATURES             Location/Qualifiers
     source          1..5966
                     /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(2591..2624)
                     /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    2645..2678
                     /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             2691..2711
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    4510..4534
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(4535..5560)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(4535..5560)
                     /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(5563..5587)
                     /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 tggcgaagag cctgcaggta accacgcgtt gcatgctctt
     2581 cggtacccgg gaagttccta tactatttga agaataggaa cttctgcagg aattcgatat
     2641 cggtgaagtt cctattctct agaaagtata ggaacttcaa aatgggatcc ccaaagaaga
     2701 agcgtaaggt gtcgatggtt tccaagggag aggccgtcat caaggagttc atgcgtttca
     2761 aggtccacat ggagggatcc atgaacggac acgagttcga gatcgaggga gagggagagg
     2821 gacgtccata cgagggaacc caaaccgcca agctcaaggt caccaaggta agtttaaaca
     2881 tatatatact aactaaccct gattatttaa attttcaggg aggaccactc ccattctcct
     2941 gggacatcct ctccccacaa ttcatgtacg gatcccgtgc cttcaccaag cacccagccg
     3001 acatcccaga ctactacaag caatccttcc cagagggatt caagtgggag cgtgtcatga
     3061 acttcgagga cggaggagcc gtcaccgtca cccaagacac ctccctcgag gacggaaccc
     3121 tcatctacaa ggtaagttta aacagttcgg tactaactaa ccatacatat ttaaattttc
     3181 aggtcaagct ccgtggaacc aacttcccac cagacggacc agtcatgcaa aagaagacca
     3241 tgggatggga ggcctccacc gagcgtctct acccagagga cggagtcctc aagggagaca
     3301 tcaagatggc cctccgtctc aaggacggag gacgttacct cgccgacttc aaggtaagtt
     3361 taaacatgat tttactaact aactaatctg atttaaattt tcagaccacc tacaaggcca
     3421 agaagccagt ccaaatgcca ggagcctaca acgtcgaccg taagctcgac atcacctccc
     3481 acaacgagga ctacaccgtc gtcgagcaat acgagcgttc cgagggacgt cactccaccg
     3541 gaggaatgga cgagctctac aaggcctccc gtcgtcgtaa ggccaaccca accaagctct
     3601 ccgagaacgc caagaagctc gccaagtaga tcaactgctc taattccatc tcgtttctct
     3661 cttttctttc aatctcttta cccgtttctc ttcccctacg tcccccaaac ctaccactgc
     3721 acagtaacac atacacattc aacgtgtcac aataaaagcc catcaacacc ttctctgttg
     3781 ttttttttct tcaaatgcat ttttttcact ttgtttaata atttttacgc atcggatttg
     3841 taggatagtg gaaatttgtc cttcaatgct tcaattgatt cgacacttct caatattttt
     3901 gatcaataaa ccattgattt tctatttaga agaagatcac tgtaaataac accaaaaaca
     3961 tcacagcaaa aaaactccca ctcaattata ttactgtttt tattgaaact agaataaatt
     4021 tgaaaatctt caaagcatac acaattaaaa aaaacgaaaa aagaaaaagt cccgttgatt
     4081 ctcaaactta aatttttaaa cttgtctgaa ccatgttgta agtttgtaaa agtttcccag
     4141 ttgtcctcga acaggtctaa aaattgcggt cataaactga aacgtaacat atgataaggt
     4201 attttgtgtg cggttttttc tatgatgttt caaaaaaatt tatcagaagt aaaaaacata
     4261 agaagtgtac acagaacatt gtgtagattt tgggatagaa ttattattaa tggggggagg
     4321 gagcacaatt tttttcgact ttttattacg aattaattct atttttgaat ctacacaatt
     4381 tcattgttag aggtgactta aaagaagcta aaaaacaaag aaattaagag aagttttttg
     4441 ataataacaa aaataggggg tgggagcaca gggagaaaga gcatgtaggg atgttgaaga
     4501 gtaattggac ccactttgta caagaaagct gggtctattc ctttgccctc ggacgagtgc
     4561 tggggcgtcg gtttccacta tcggcgagta cttctacaca gccatcggtc cagacggccg
     4621 cgcttctgcg ggcgatttgt gtacgcccga cagtcccggc tccggatcgg acgattgcgt
     4681 cgcatcgacc ctgcgcccaa gctgcatcat cgaaattgcc gtcaaccaag ctctgataga
     4741 gttggtcaag accaatgcgg agcatatacg cccggagccg cggcgatcct gcaagctccg
     4801 gatgcctccg ctcgaagtag cgcgtctgct gctccataca agccaaccac ggcctccaga
     4861 agaagatgtt ggcgacctcg tattgggaat ccccgaacat cgcctcgctc cagtcaatga
     4921 ccgctgttat gcggccattg tccgtcagga cattgttgga gccgaaatcc gcgtgcacga
     4981 ggtgccggac ttcggggcag tcctcggccc aaagcatcag ctcatcgaga gcctgcgcga
     5041 cggacgcact gacggtgtcg tccatcacag tttgccagtg atacacatgg ggatcagcaa
     5101 tcgcgcatat gaaatcacgc catgtagtgt attgaccgat tccttgcggt ccgaatgggc
     5161 cgaacccgct cgtctggcta agatcggccg cagcgatcgc atccatggcc tccgcgaccg
     5221 gctgcagaac agcgggcagt tcggtttcag gcaggtcttg caacgtgaca ccctgtgcac
     5281 ggcgggagat gcaataggtc aggctctcgc tgaattcccc aatgtcaagc acttccggaa
     5341 tcgggagcgc ggccgatgca aagtgccgat aaacataacg atctttgtag aaaccatcgg
     5401 cgcagctatt tacccgcagg acatatccac gccctcctac atcgaagctg aaagcacgag
     5461 attcttcgcc ctccgagagc tgcatcaggt cggagacgct gtcgaacttt tcgatcagaa
     5521 acttctcgac agacgtcgcg gtgagttcag gctttttcat ggagcctgct tttttgtaca
     5581 aacttgcggc accgcctgac atattacctt aaaattcaaa aattaatttc agatcagtca
     5641 taaccaagaa aacaaaataa acgttaggac agaaaattca aattaattaa aaattatgct
     5701 gaataagaaa attacatgca aagccttatt ttcctcatag aaacagttga ataacatatt
     5761 ttttaattga aaacatagca aaattcgtgg taaaatttgt caaaaccttg gaaaaaatcg
     5821 aaaaaagaac atttcccgat gcattttcct atttctcgtt gccgacaacg tataaacgtt
     5881 gaatactcag tcaacgaatt tggctgctgg cttggactct cgcacggaag agacgcgtgt
     5941 gtagatttac gacgaaagca aaaatg
//
