LOCUS       sequence-393491-        6005 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  hygR selection nls-mNG marked rapid RMCE integration vector without 
            loxP site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6005)
  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 6005)
  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: pHygG8
FEATURES             Location/Qualifiers
     source          1..6005
                     /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(2567..2600)
                     /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    2621..2654
                     /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             2667..2687
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    4549..4573
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(4574..5599)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(4574..5599)
                     /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(5602..5626)
                     /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 ggagctccac cgcggtggcg gccgctctag aactagtggc
     2521 gaagagcctg caggtaacca cgcgttgcat gctcttcggt acccgggaag ttcctatact
     2581 atttgaagaa taggaacttc tgcaggaatt cgatatcggt gaagttccta ttctctagaa
     2641 agtataggaa cttcaaaatg ggatccccaa agaagaagcg taaggtgtcg atggtgtcga
     2701 agggagaaga ggataacatg gcttcactcc cagctacaca cgaactccac atcttcggat
     2761 cgatcaacgg agtggatttc gatatggtcg gacaaggtaa gtttaaacat atatatacta
     2821 actaaccctg attatttaaa ttttcaggaa ctggaaaccc aaacgatgga tacgaggaac
     2881 tcaacctcaa gtcgacaaag ggagatctgc aattctcgcc atggattctc gtgccacaca
     2941 tcggatacgg attccaccaa tacctcccat acccaggtaa gtttaaactg agttctacta
     3001 actaacgagt aatatttaaa ttttcagatg gaatgtcacc attccaagct gccatggtgg
     3061 atggatcggg ataccaagtt caccgaacaa tgcaattcga ggatggagcc tcgctcacag
     3121 tgaactaccg atacacatac gagggatcgc acatcaaggt aagtttaaac agttcggtac
     3181 taactaacca tacatattta aattttcagg gagaggctca agttaaggga acaggattcc
     3241 cagctgatgg accagtgatg acaaactcac tcacagctgc tgattggtgc cgatcgaaaa
     3301 agacataccc aaatgataag acaatcatct cgacattcaa gtggtcgtac actactggaa
     3361 acggaaagcg ataccgatcg acagcccgaa caacatacac attcgctaag ccaatggccg
     3421 ccaactacct caaggtaagt ttaaacatga ttttactaac taactaatct gatttaaatt
     3481 ttcagaatca accaatgtac gtgttccgaa agacagaact caagcactca aagacagagc
     3541 tgaacttcaa agagtggcaa aaggccttca cagatgtgat gggaatggat gaactctaca
     3601 aggcctcccg tcgtcgtaag gccaacccaa ccaagctctc cgagaacgcc aagaagctcg
     3661 ccaagtagat caactgctct aattccatct cgtttctctc ttttctttca atctctttac
     3721 ccgtttctct tcccctacgt cccccaaacc taccactgca cagtaacaca tacacattca
     3781 acgtgtcaca ataaaagccc atcaacacct tctctgttgt tttttttctt caaatgcatt
     3841 tttttcactt tgtttaataa tttttacgca tcggatttgt aggatagtgg aaatttgtcc
     3901 ttcaatgctt caattgattc gacacttctc aatatttttg atcaataaac cattgatttt
     3961 ctatttagaa gaagatcact gtaaataaca ccaaaaacat cacagcaaaa aaactcccac
     4021 tcaattatat tactgttttt attgaaacta gaataaattt gaaaatcttc aaagcataca
     4081 caattaaaaa aaacgaaaaa agaaaaagtc ccgttgattc tcaaacttaa atttttaaac
     4141 ttgtctgaac catgttgtaa gtttgtaaaa gtttcccagt tgtcctcgaa caggtctaaa
     4201 aattgcggtc ataaactgaa acgtaacata tgataaggta ttttgtgtgc ggttttttct
     4261 atgatgtttc aaaaaaattt atcagaagta aaaaacataa gaagtgtaca cagaacattg
     4321 tgtagatttt gggatagaat tattattaat ggggggaggg agcacaattt ttttcgactt
     4381 tttattacga attaattcta tttttgaatc tacacaattt cattgttaga ggtgacttaa
     4441 aagaagctaa aaaacaaaga aattaagaga agttttttga taataacaaa aatagggggt
     4501 gggagcacag ggagaaagag catgtaggga tgttgaagag taattggacc cactttgtac
     4561 aagaaagctg ggtctattcc tttgccctcg gacgagtgct ggggcgtcgg tttccactat
     4621 cggcgagtac ttctacacag ccatcggtcc agacggccgc gcttctgcgg gcgatttgtg
     4681 tacgcccgac agtcccggct ccggatcgga cgattgcgtc gcatcgaccc tgcgcccaag
     4741 ctgcatcatc gaaattgccg tcaaccaagc tctgatagag ttggtcaaga ccaatgcgga
     4801 gcatatacgc ccggagccgc ggcgatcctg caagctccgg atgcctccgc tcgaagtagc
     4861 gcgtctgctg ctccatacaa gccaaccacg gcctccagaa gaagatgttg gcgacctcgt
     4921 attgggaatc cccgaacatc gcctcgctcc agtcaatgac cgctgttatg cggccattgt
     4981 ccgtcaggac attgttggag ccgaaatccg cgtgcacgag gtgccggact tcggggcagt
     5041 cctcggccca aagcatcagc tcatcgagag cctgcgcgac ggacgcactg acggtgtcgt
     5101 ccatcacagt ttgccagtga tacacatggg gatcagcaat cgcgcatatg aaatcacgcc
     5161 atgtagtgta ttgaccgatt ccttgcggtc cgaatgggcc gaacccgctc gtctggctaa
     5221 gatcggccgc agcgatcgca tccatggcct ccgcgaccgg ctgcagaaca gcgggcagtt
     5281 cggtttcagg caggtcttgc aacgtgacac cctgtgcacg gcgggagatg caataggtca
     5341 ggctctcgct gaattcccca atgtcaagca cttccggaat cgggagcgcg gccgatgcaa
     5401 agtgccgata aacataacga tctttgtaga aaccatcggc gcagctattt acccgcagga
     5461 catatccacg ccctcctaca tcgaagctga aagcacgaga ttcttcgccc tccgagagct
     5521 gcatcaggtc ggagacgctg tcgaactttt cgatcagaaa cttctcgaca gacgtcgcgg
     5581 tgagttcagg ctttttcatg gagcctgctt ttttgtacaa acttgcggca ccgcctgaca
     5641 tattacctta aaattcaaaa attaatttca gatcagtcat aaccaagaaa acaaaataaa
     5701 cgttaggaca gaaaattcaa attaattaaa aattatgctg aataagaaaa ttacatgcaa
     5761 agccttattt tcctcataga aacagttgaa taacatattt tttaattgaa aacatagcaa
     5821 aattcgtggt aaaatttgtc aaaaccttgg aaaaaatcga aaaaagaaca tttcccgatg
     5881 cattttccta tttctcgttg ccgacaacgt ataaacgttg aatactcagt caacgaattt
     5941 ggctgctggc ttggactctc gcacggaaga gacgcgtgtg tagatttacg acgaaagcaa
     6001 aaatg
//
