LOCUS       sequence-393497-        5981 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  hygR selection GFP marked rapid RMCE integration vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5981)
  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 5981)
  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: pHygG6
FEATURES             Location/Qualifiers
     source          1..5981
                     /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    4525..4549
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(4550..5575)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(4550..5575)
                     /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(5578..5602)
                     /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 gtcgatgagt aaaggagaag aattgttcac tggagttgtc ccaatcctcg
     2761 tcgagctcga cggagacgtc aacggacaca agttctccgt ctccggagag ggagagggag
     2821 acgccaccta cggaaagctc accctcaagt tcatctgcac caccggaaag ctcccagtcc
     2881 catggccaac cctcgtcacc accttctgct acggagtcca atgcttctcc cgttacccag
     2941 accacatgaa gcgtcacgac ttcttcaagt ccgccatgcc agagggatac gtccaagagc
     3001 gtaccatctt cttcaaggta agtttaaaca tatatatact aactactgat tatttaaatt
     3061 ttcaggacga cggaaactac aagacccgtg ccgaggtcaa gttcgaggga gacaccctcg
     3121 tcaaccgtat cgagctcaag gtaagtttaa acagttcggt actaactaac catacatatt
     3181 taaattttca gggaatcgac ttcaaggagg acggaaacat cctcggacac aagctcgagt
     3241 acaactacaa ctcccacaac gtctacatca tggccgacaa gcaaaagaac ggaatcaagg
     3301 tcaacttcaa ggtaagttta aacatgattt tactaactaa ctaatctgat ttaaattttc
     3361 agatccgtca caacatcgag gacggatccg tccaactcgc cgaccactac caacaaaaca
     3421 ccccaatcgg agacggacca gtcctcctcc cagacaacca ctacctctcc acccaatccg
     3481 ccctctccaa ggacccaaac gagaagcgtg accacatggt cctcctcgag ttcgtcaccg
     3541 ccgccggaat cacccacgga atggacgagc tctacaaggc ctcccgtcgt cgtaaggcca
     3601 acccaaccaa gctctccgag aacgccaaga agctcgccaa gtagatcaac tgctctaatt
     3661 ccatctcgtt tctctctttt ctttcaatct ctttacccgt ttctcttccc ctacgtcccc
     3721 caaacctacc actgcacagt aacacataca cattcaacgt gtcacaataa aagcccatca
     3781 acaccttctc tgttgttttt tttcttcaaa tgcatttttt tcactttgtt taataatttt
     3841 tacgcatcgg atttgtagga tagtggaaat ttgtccttca atgcttcaat tgattcgaca
     3901 cttctcaata tttttgatca ataaaccatt gattttctat ttagaagaag atcactgtaa
     3961 ataacaccaa aaacatcaca gcaaaaaaac tcccactcaa ttatattact gtttttattg
     4021 aaactagaat aaatttgaaa atcttcaaag catacacaat taaaaaaaac gaaaaaagaa
     4081 aaagtcccgt tgattctcaa acttaaattt ttaaacttgt ctgaaccatg ttgtaagttt
     4141 gtaaaagttt cccagttgtc ctcgaacagg tctaaaaatt gcggtcataa actgaaacgt
     4201 aacatatgat aaggtatttt gtgtgcggtt ttttctatga tgtttcaaaa aaatttatca
     4261 gaagtaaaaa acataagaag tgtacacaga acattgtgta gattttggga tagaattatt
     4321 attaatgggg ggagggagca caattttttt cgacttttta ttacgaatta attctatttt
     4381 tgaatctaca caatttcatt gttagaggtg acttaaaaga agctaaaaaa caaagaaatt
     4441 aagagaagtt ttttgataat aacaaaaata gggggtggga gcacagggag aaagagcatg
     4501 tagggatgtt gaagagtaat tggacccact ttgtacaaga aagctgggtc tattcctttg
     4561 ccctcggacg agtgctgggg cgtcggtttc cactatcggc gagtacttct acacagccat
     4621 cggtccagac ggccgcgctt ctgcgggcga tttgtgtacg cccgacagtc ccggctccgg
     4681 atcggacgat tgcgtcgcat cgaccctgcg cccaagctgc atcatcgaaa ttgccgtcaa
     4741 ccaagctctg atagagttgg tcaagaccaa tgcggagcat atacgcccgg agccgcggcg
     4801 atcctgcaag ctccggatgc ctccgctcga agtagcgcgt ctgctgctcc atacaagcca
     4861 accacggcct ccagaagaag atgttggcga cctcgtattg ggaatccccg aacatcgcct
     4921 cgctccagtc aatgaccgct gttatgcggc cattgtccgt caggacattg ttggagccga
     4981 aatccgcgtg cacgaggtgc cggacttcgg ggcagtcctc ggcccaaagc atcagctcat
     5041 cgagagcctg cgcgacggac gcactgacgg tgtcgtccat cacagtttgc cagtgataca
     5101 catggggatc agcaatcgcg catatgaaat cacgccatgt agtgtattga ccgattcctt
     5161 gcggtccgaa tgggccgaac ccgctcgtct ggctaagatc ggccgcagcg atcgcatcca
     5221 tggcctccgc gaccggctgc agaacagcgg gcagttcggt ttcaggcagg tcttgcaacg
     5281 tgacaccctg tgcacggcgg gagatgcaat aggtcaggct ctcgctgaat tccccaatgt
     5341 caagcacttc cggaatcggg agcgcggccg atgcaaagtg ccgataaaca taacgatctt
     5401 tgtagaaacc atcggcgcag ctatttaccc gcaggacata tccacgccct cctacatcga
     5461 agctgaaagc acgagattct tcgccctccg agagctgcat caggtcggag acgctgtcga
     5521 acttttcgat cagaaacttc tcgacagacg tcgcggtgag ttcaggcttt ttcatggagc
     5581 ctgctttttt gtacaaactt gcggcaccgc ctgacatatt accttaaaat tcaaaaatta
     5641 atttcagatc agtcataacc aagaaaacaa aataaacgtt aggacagaaa attcaaatta
     5701 attaaaaatt atgctgaata agaaaattac atgcaaagcc ttattttcct catagaaaca
     5761 gttgaataac atatttttta attgaaaaca tagcaaaatt cgtggtaaaa tttgtcaaaa
     5821 ccttggaaaa aatcgaaaaa agaacatttc ccgatgcatt ttcctatttc tcgttgccga
     5881 caacgtataa acgttgaata ctcagtcaac gaatttggct gctggcttgg actctcgcac
     5941 ggaagagacg cgtgtgtaga tttacgacga aagcaaaaat g
//
