LOCUS       sequence-393495-        6029 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  hygR selection nls-mNG marked rapid RMCE integration vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6029)
  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 6029)
  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: pHygG2
FEATURES             Location/Qualifiers
     source          1..6029
                     /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    4573..4597
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(4598..5623)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(4598..5623)
                     /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(5626..5650)
                     /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 gtcgatggtg tcgaagggag aagaggataa catggcttca ctcccagcta
     2761 cacacgaact ccacatcttc ggatcgatca acggagtgga tttcgatatg gtcggacaag
     2821 gtaagtttaa acatatatat actaactaac cctgattatt taaattttca ggaactggaa
     2881 acccaaacga tggatacgag gaactcaacc tcaagtcgac aaagggagat ctgcaattct
     2941 cgccatggat tctcgtgcca cacatcggat acggattcca ccaatacctc ccatacccag
     3001 gtaagtttaa actgagttct actaactaac gagtaatatt taaattttca gatggaatgt
     3061 caccattcca agctgccatg gtggatggat cgggatacca agttcaccga acaatgcaat
     3121 tcgaggatgg agcctcgctc acagtgaact accgatacac atacgaggga tcgcacatca
     3181 aggtaagttt aaacagttcg gtactaacta accatacata tttaaatttt cagggagagg
     3241 ctcaagttaa gggaacagga ttcccagctg atggaccagt gatgacaaac tcactcacag
     3301 ctgctgattg gtgccgatcg aaaaagacat acccaaatga taagacaatc atctcgacat
     3361 tcaagtggtc gtacactact ggaaacggaa agcgataccg atcgacagcc cgaacaacat
     3421 acacattcgc taagccaatg gccgccaact acctcaaggt aagtttaaac atgattttac
     3481 taactaacta atctgattta aattttcaga atcaaccaat gtacgtgttc cgaaagacag
     3541 aactcaagca ctcaaagaca gagctgaact tcaaagagtg gcaaaaggcc ttcacagatg
     3601 tgatgggaat ggatgaactc tacaaggcct cccgtcgtcg taaggccaac ccaaccaagc
     3661 tctccgagaa cgccaagaag ctcgccaagt agatcaactg ctctaattcc atctcgtttc
     3721 tctcttttct ttcaatctct ttacccgttt ctcttcccct acgtccccca aacctaccac
     3781 tgcacagtaa cacatacaca ttcaacgtgt cacaataaaa gcccatcaac accttctctg
     3841 ttgttttttt tcttcaaatg catttttttc actttgttta ataattttta cgcatcggat
     3901 ttgtaggata gtggaaattt gtccttcaat gcttcaattg attcgacact tctcaatatt
     3961 tttgatcaat aaaccattga ttttctattt agaagaagat cactgtaaat aacaccaaaa
     4021 acatcacagc aaaaaaactc ccactcaatt atattactgt ttttattgaa actagaataa
     4081 atttgaaaat cttcaaagca tacacaatta aaaaaaacga aaaaagaaaa agtcccgttg
     4141 attctcaaac ttaaattttt aaacttgtct gaaccatgtt gtaagtttgt aaaagtttcc
     4201 cagttgtcct cgaacaggtc taaaaattgc ggtcataaac tgaaacgtaa catatgataa
     4261 ggtattttgt gtgcggtttt ttctatgatg tttcaaaaaa atttatcaga agtaaaaaac
     4321 ataagaagtg tacacagaac attgtgtaga ttttgggata gaattattat taatgggggg
     4381 agggagcaca atttttttcg actttttatt acgaattaat tctatttttg aatctacaca
     4441 atttcattgt tagaggtgac ttaaaagaag ctaaaaaaca aagaaattaa gagaagtttt
     4501 ttgataataa caaaaatagg gggtgggagc acagggagaa agagcatgta gggatgttga
     4561 agagtaattg gacccacttt gtacaagaaa gctgggtcta ttcctttgcc ctcggacgag
     4621 tgctggggcg tcggtttcca ctatcggcga gtacttctac acagccatcg gtccagacgg
     4681 ccgcgcttct gcgggcgatt tgtgtacgcc cgacagtccc ggctccggat cggacgattg
     4741 cgtcgcatcg accctgcgcc caagctgcat catcgaaatt gccgtcaacc aagctctgat
     4801 agagttggtc aagaccaatg cggagcatat acgcccggag ccgcggcgat cctgcaagct
     4861 ccggatgcct ccgctcgaag tagcgcgtct gctgctccat acaagccaac cacggcctcc
     4921 agaagaagat gttggcgacc tcgtattggg aatccccgaa catcgcctcg ctccagtcaa
     4981 tgaccgctgt tatgcggcca ttgtccgtca ggacattgtt ggagccgaaa tccgcgtgca
     5041 cgaggtgccg gacttcgggg cagtcctcgg cccaaagcat cagctcatcg agagcctgcg
     5101 cgacggacgc actgacggtg tcgtccatca cagtttgcca gtgatacaca tggggatcag
     5161 caatcgcgca tatgaaatca cgccatgtag tgtattgacc gattccttgc ggtccgaatg
     5221 ggccgaaccc gctcgtctgg ctaagatcgg ccgcagcgat cgcatccatg gcctccgcga
     5281 ccggctgcag aacagcgggc agttcggttt caggcaggtc ttgcaacgtg acaccctgtg
     5341 cacggcggga gatgcaatag gtcaggctct cgctgaattc cccaatgtca agcacttccg
     5401 gaatcgggag cgcggccgat gcaaagtgcc gataaacata acgatctttg tagaaaccat
     5461 cggcgcagct atttacccgc aggacatatc cacgccctcc tacatcgaag ctgaaagcac
     5521 gagattcttc gccctccgag agctgcatca ggtcggagac gctgtcgaac ttttcgatca
     5581 gaaacttctc gacagacgtc gcggtgagtt caggcttttt catggagcct gcttttttgt
     5641 acaaacttgc ggcaccgcct gacatattac cttaaaattc aaaaattaat ttcagatcag
     5701 tcataaccaa gaaaacaaaa taaacgttag gacagaaaat tcaaattaat taaaaattat
     5761 gctgaataag aaaattacat gcaaagcctt attttcctca tagaaacagt tgaataacat
     5821 attttttaat tgaaaacata gcaaaattcg tggtaaaatt tgtcaaaacc ttggaaaaaa
     5881 tcgaaaaaag aacatttccc gatgcatttt cctatttctc gttgccgaca acgtataaac
     5941 gttgaatact cagtcaacga atttggctgc tggcttggac tctcgcacgg aagagacgcg
     6001 tgtgtagatt tacgacgaaa gcaaaaatg
//
