LOCUS       sequence-393486-        4535 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  hygR selection unmarked rapid RMCE integration vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4535)
  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 4535)
  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: pHygO2r
FEATURES             Location/Qualifiers
     source          1..4535
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    379..403
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(404..1429)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(404..1429)
                     /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(1432..1456)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1935..1952)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2106..2694)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2186..2205)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2865..3656,3657..3725))
                     /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     3488..3507
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3726..3830)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3856..4311)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3993..4014)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4205..4224
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    complement(4323..4356)
                     /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(4436..4469)
                     /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    4490..4523
                     /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)"
ORIGIN
        1 taggcccagt tgtcctcgaa caggtctaaa aattgcggtc ataaactgaa acgtaacata
       61 tgataaggta ttttgtgtgc ggttttttct atgatgtttc aaaaaaattt atcagaagta
      121 aaaaacataa gaagtgtaca cagaacattg tgtagatttt gggatagaat tattattaat
      181 ggggggaggg agcacaattt ttttcgactt tttattacga attaattcta tttttgaatc
      241 tacacaattt cattgttaga ggtgacttaa aagaagctaa aaaacaaaga aattaagaga
      301 agttttttga taataacaaa aatagggggt gggagcacag ggagaaagag catgtaggga
      361 tgttgaagag taattggacc cactttgtac aagaaagctg ggtctattcc tttgccctcg
      421 gacgagtgct ggggcgtcgg tttccactat cggcgagtac ttctacacag ccatcggtcc
      481 agacggccgc gcttctgcgg gcgatttgtg tacgcccgac agtcccggct ccggatcgga
      541 cgattgcgtc gcatcgaccc tgcgcccaag ctgcatcatc gaaattgccg tcaaccaagc
      601 tctgatagag ttggtcaaga ccaatgcgga gcatatacgc ccggagccgc ggcgatcctg
      661 caagctccgg atgcctccgc tcgaagtagc gcgtctgctg ctccatacaa gccaaccacg
      721 gcctccagaa gaagatgttg gcgacctcgt attgggaatc cccgaacatc gcctcgctcc
      781 agtcaatgac cgctgttatg cggccattgt ccgtcaggac attgttggag ccgaaatccg
      841 cgtgcacgag gtgccggact tcggggcagt cctcggccca aagcatcagc tcatcgagag
      901 cctgcgcgac ggacgcactg acggtgtcgt ccatcacagt ttgccagtga tacacatggg
      961 gatcagcaat cgcgcatatg aaatcacgcc atgtagtgta ttgaccgatt ccttgcggtc
     1021 cgaatgggcc gaacccgctc gtctggctaa gatcggccgc agcgatcgca tccatggcct
     1081 ccgcgaccgg ctgcagaaca gcgggcagtt cggtttcagg caggtcttgc aacgtgacac
     1141 cctgtgcacg gcgggagatg caataggtca ggctctcgct gaattcccca atgtcaagca
     1201 cttccggaat cgggagcgcg gccgatgcaa agtgccgata aacataacga tctttgtaga
     1261 aaccatcggc gcagctattt acccgcagga catatccacg ccctcctaca tcgaagctga
     1321 aagcacgaga ttcttcgccc tccgagagct gcatcaggtc ggagacgctg tcgaactttt
     1381 cgatcagaaa cttctcgaca gacgtcgcgg tgagttcagg ctttttcatg gagcctgctt
     1441 ttttgtacaa acttgcggca ccgcctgaca tattacctta aaattcaaaa attaatttca
     1501 gatcagtcat aaccaagaaa acaaaataaa cgttaggaca gaaaattcaa attaattaaa
     1561 aattatgctg aataagaaaa ttacatgcaa agccttattt tcctcataga aacagttgaa
     1621 taacatattt tttaattgaa aacatagcaa aattcgtggt aaaatttgtc aaaaccttgg
     1681 aaaaaatcga aaaaagaaca tttcccgatg cattttccta tttctcgttg ccgacaacgt
     1741 ataaacgttg aatactcagt caacgaattt ggctgctggc ttggactctc gcacggaaga
     1801 gacgcgtgtg tagatttacg acgaaagcaa aaatgatccc gctttccagt cgggaaacct
     1861 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     1921 gcgaattccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     1981 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     2041 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     2101 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     2161 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     2221 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     2281 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     2341 cgctccaagc tgggctgtgt gcacgaaccc cccattcagc ccgaccgctg cgccttatcc
     2401 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     2461 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     2521 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     2581 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     2641 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     2701 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     2761 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     2821 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     2881 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     2941 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     3001 ccgcgagatc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     3061 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     3121 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     3181 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     3241 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     3301 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     3361 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     3421 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     3481 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     3541 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     3601 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     3661 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     3721 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     3781 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     3841 cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     3901 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     3961 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     4021 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     4081 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     4141 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     4201 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     4261 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcgtcggagc
     4321 tcataacttc gtatagcata cattatacga agttatgcgg ccgctctaga actagtggta
     4381 ccgaagagca tgcaacgcgt ggttacctgc aggctcttcg ccacttaaga cccgggaagt
     4441 tcctatacta tttgaagaat aggaacttct gcaggaattc gatatcggtg aagttcctat
     4501 tctctagaaa gtataggaac ttcaaaatgg gatcc
//
