LOCUS       sequence-393490-        4525 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 4525)
  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 4525)
  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: pHygO2
FEATURES             Location/Qualifiers
     source          1..4525
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    385..409
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(410..1435)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(410..1435)
                     /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(1438..1462)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1941..1958)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2112..2700)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2192..2211)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2871..3662,3663..3731))
                     /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     3494..3513
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3732..3836)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3862..4317)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3999..4020)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4211..4230
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    complement(4329..4362)
                     /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(4432..4465)
                     /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    4486..4519
                     /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 ggatcctagg cccagttgtc ctcgaacagg tctaaaaatt gcggtcataa actgaaacgt
       61 aacatatgat aaggtatttt gtgtgcggtt ttttctatga tgtttcaaaa aaatttatca
      121 gaagtaaaaa acataagaag tgtacacaga acattgtgta gattttggga tagaattatt
      181 attaatgggg ggagggagca caattttttt cgacttttta ttacgaatta attctatttt
      241 tgaatctaca caatttcatt gttagaggtg acttaaaaga agctaaaaaa caaagaaatt
      301 aagagaagtt ttttgataat aacaaaaata gggggtggga gcacagggag aaagagcatg
      361 tagggatgtt gaagagtaat tggacccact ttgtacaaga aagctgggtc tattcctttg
      421 ccctcggacg agtgctgggg cgtcggtttc cactatcggc gagtacttct acacagccat
      481 cggtccagac ggccgcgctt ctgcgggcga tttgtgtacg cccgacagtc ccggctccgg
      541 atcggacgat tgcgtcgcat cgaccctgcg cccaagctgc atcatcgaaa ttgccgtcaa
      601 ccaagctctg atagagttgg tcaagaccaa tgcggagcat atacgcccgg agccgcggcg
      661 atcctgcaag ctccggatgc ctccgctcga agtagcgcgt ctgctgctcc atacaagcca
      721 accacggcct ccagaagaag atgttggcga cctcgtattg ggaatccccg aacatcgcct
      781 cgctccagtc aatgaccgct gttatgcggc cattgtccgt caggacattg ttggagccga
      841 aatccgcgtg cacgaggtgc cggacttcgg ggcagtcctc ggcccaaagc atcagctcat
      901 cgagagcctg cgcgacggac gcactgacgg tgtcgtccat cacagtttgc cagtgataca
      961 catggggatc agcaatcgcg catatgaaat cacgccatgt agtgtattga ccgattcctt
     1021 gcggtccgaa tgggccgaac ccgctcgtct ggctaagatc ggccgcagcg atcgcatcca
     1081 tggcctccgc gaccggctgc agaacagcgg gcagttcggt ttcaggcagg tcttgcaacg
     1141 tgacaccctg tgcacggcgg gagatgcaat aggtcaggct ctcgctgaat tccccaatgt
     1201 caagcacttc cggaatcggg agcgcggccg atgcaaagtg ccgataaaca taacgatctt
     1261 tgtagaaacc atcggcgcag ctatttaccc gcaggacata tccacgccct cctacatcga
     1321 agctgaaagc acgagattct tcgccctccg agagctgcat caggtcggag acgctgtcga
     1381 acttttcgat cagaaacttc tcgacagacg tcgcggtgag ttcaggcttt ttcatggagc
     1441 ctgctttttt gtacaaactt gcggcaccgc ctgacatatt accttaaaat tcaaaaatta
     1501 atttcagatc agtcataacc aagaaaacaa aataaacgtt aggacagaaa attcaaatta
     1561 attaaaaatt atgctgaata agaaaattac atgcaaagcc ttattttcct catagaaaca
     1621 gttgaataac atatttttta attgaaaaca tagcaaaatt cgtggtaaaa tttgtcaaaa
     1681 ccttggaaaa aatcgaaaaa agaacatttc ccgatgcatt ttcctatttc tcgttgccga
     1741 caacgtataa acgttgaata ctcagtcaac gaatttggct gctggcttgg actctcgcac
     1801 ggaagagacg cgtgtgtaga tttacgacga aagcaaaaat gatcccgctt tccagtcggg
     1861 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     1921 tattgggcga attccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     1981 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     2041 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     2101 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     2161 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     2221 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     2281 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     2341 gtcgttcgct ccaagctggg ctgtgtgcac gaacccccca ttcagcccga ccgctgcgcc
     2401 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     2461 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     2521 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     2581 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     2641 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     2701 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     2761 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     2821 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     2881 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     2941 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     3001 atgataccgc gagatccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     3061 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     3121 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     3181 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     3241 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     3301 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     3361 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     3421 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     3481 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     3541 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     3601 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     3661 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     3721 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     3781 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     3841 tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     3901 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     3961 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     4021 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     4081 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     4141 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     4201 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     4261 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcgt
     4321 cggagctcat aacttcgtat agcatacatt atacgaagtt atgcggccgc tctagaacta
     4381 gtggcgaaga gcctgcaggt aaccacgcgt tgcatgctct tcggtacccg ggaagttcct
     4441 atactatttg aagaatagga acttctgcag gaattcgata tcggtgaagt tcctattctc
     4501 tagaaagtat aggaacttca aaatg
//
