LOCUS       sequence-393492-        5763 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  hygR selection mNG marked rapid RMCE integration vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5763)
  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 5763)
  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: pHygCG2r
FEATURES             Location/Qualifiers
     source          1..5763
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    375..399
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(400..1425)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(400..1425)
                     /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(1428..1452)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1931..1948)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2102..2690)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2182..2201)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2861..3652,3653..3721))
                     /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     3484..3503
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3722..3826)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3852..4307)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3989..4010)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4201..4220
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    complement(4319..4352)
                     /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)"
     terminator      join(5441..5763,1)
                     /gene="tbb-2"
                     /label=tbb-2 terminator
                     /note="transcription terminator for the C. elegans tubulin 
                     beta-2 gene"
ORIGIN
        1 ctcagttgtc ctcgaacagg tctaaaaatt gcggtcataa actgaaacgt aacatatgat
       61 aaggtatttt gtgtgcggtt ttttctatga tgtttcaaaa aaatttatca gaagtaaaaa
      121 acataagaag tgtacacaga acattgtgta gattttggga tagaattatt attaatgggg
      181 ggagggagca caattttttt cgacttttta ttacgaatta attctatttt tgaatctaca
      241 caatttcatt gttagaggtg acttaaaaga agctaaaaaa caaagaaatt aagagaagtt
      301 ttttgataat aacaaaaata gggggtggga gcacagggag aaagagcatg tagggatgtt
      361 gaagagtaat tggacccact ttgtacaaga aagctgggtc tattcctttg ccctcggacg
      421 agtgctgggg cgtcggtttc cactatcggc gagtacttct acacagccat cggtccagac
      481 ggccgcgctt ctgcgggcga tttgtgtacg cccgacagtc ccggctccgg atcggacgat
      541 tgcgtcgcat cgaccctgcg cccaagctgc atcatcgaaa ttgccgtcaa ccaagctctg
      601 atagagttgg tcaagaccaa tgcggagcat atacgcccgg agccgcggcg atcctgcaag
      661 ctccggatgc ctccgctcga agtagcgcgt ctgctgctcc atacaagcca accacggcct
      721 ccagaagaag atgttggcga cctcgtattg ggaatccccg aacatcgcct cgctccagtc
      781 aatgaccgct gttatgcggc cattgtccgt caggacattg ttggagccga aatccgcgtg
      841 cacgaggtgc cggacttcgg ggcagtcctc ggcccaaagc atcagctcat cgagagcctg
      901 cgcgacggac gcactgacgg tgtcgtccat cacagtttgc cagtgataca catggggatc
      961 agcaatcgcg catatgaaat cacgccatgt agtgtattga ccgattcctt gcggtccgaa
     1021 tgggccgaac ccgctcgtct ggctaagatc ggccgcagcg atcgcatcca tggcctccgc
     1081 gaccggctgc agaacagcgg gcagttcggt ttcaggcagg tcttgcaacg tgacaccctg
     1141 tgcacggcgg gagatgcaat aggtcaggct ctcgctgaat tccccaatgt caagcacttc
     1201 cggaatcggg agcgcggccg atgcaaagtg ccgataaaca taacgatctt tgtagaaacc
     1261 atcggcgcag ctatttaccc gcaggacata tccacgccct cctacatcga agctgaaagc
     1321 acgagattct tcgccctccg agagctgcat caggtcggag acgctgtcga acttttcgat
     1381 cagaaacttc tcgacagacg tcgcggtgag ttcaggcttt ttcatggagc ctgctttttt
     1441 gtacaaactt gcggcaccgc ctgacatatt accttaaaat tcaaaaatta atttcagatc
     1501 agtcataacc aagaaaacaa aataaacgtt aggacagaaa attcaaatta attaaaaatt
     1561 atgctgaata agaaaattac atgcaaagcc ttattttcct catagaaaca gttgaataac
     1621 atatttttta attgaaaaca tagcaaaatt cgtggtaaaa tttgtcaaaa ccttggaaaa
     1681 aatcgaaaaa agaacatttc ccgatgcatt ttcctatttc tcgttgccga caacgtataa
     1741 acgttgaata ctcagtcaac gaatttggct gctggcttgg actctcgcac ggaagagacg
     1801 cgtgtgtaga tttacgacga aagcaaaaat gatcccgctt tccagtcggg aaacctgtcg
     1861 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcga
     1921 attccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     1981 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     2041 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     2101 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     2161 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     2221 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     2281 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     2341 ccaagctggg ctgtgtgcac gaacccccca ttcagcccga ccgctgcgcc ttatccggta
     2401 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     2461 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     2521 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     2581 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     2641 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     2701 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     2761 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     2821 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     2881 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     2941 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     3001 gagatccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     3061 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     3121 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     3181 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     3241 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     3301 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     3361 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     3421 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     3481 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     3541 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     3601 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     3661 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     3721 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     3781 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     3841 aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     3901 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     3961 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     4021 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     4081 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     4141 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     4201 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     4261 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcgt cggagctcat
     4321 aacttcgtat agcatacatt atacgaagtt atgcggccgc tctagaacta gtggtaccga
     4381 agagcatgca acgcgtggtt acctgcaggc tcttcgccac ttaagacccg ggaagttcct
     4441 atactatttg aagaatagga acttctgcag gaattcgata tcggtgaagt tcctattctc
     4501 tagaaagtat aggaacttca aaatggtgtc gaagggagaa gaggataaca tggcttcact
     4561 cccagctaca cacgaactcc acatcttcgg atcgatcaac ggagtggatt tcgatatggt
     4621 cggacaaggt aagtttaaac atatatatac taactaaccc tgattattta aattttcagg
     4681 aactggaaac ccaaacgatg gatacgagga actcaacctc aagtcgacaa agggagatct
     4741 gcaattctcg ccatggattc tcgtgccaca catcggatac ggattccacc aatacctccc
     4801 atacccaggt aagtttaaac tgagttctac taactaacga gtaatattta aattttcaga
     4861 tggaatgtca ccattccaag ctgccatggt ggatggatcg ggataccaag ttcaccgaac
     4921 aatgcaattc gaggatggag cctcgctcac agtgaactac cgatacacat acgagggatc
     4981 gcacatcaag gtaagtttaa acagttcggt actaactaac catacatatt taaattttca
     5041 gggagaggct caagttaagg gaacaggatt cccagctgat ggaccagtga tgacaaactc
     5101 actcacagct gctgattggt gccgatcgaa aaagacatac ccaaatgata agacaatcat
     5161 ctcgacattc aagtggtcgt acactactgg aaacggaaag cgataccgat cgacagcccg
     5221 aacaacatac acattcgcta agccaatggc cgccaactac ctcaaggtaa gtttaaacat
     5281 gattttacta actaactaat ctgatttaaa ttttcagaat caaccaatgt acgtgttccg
     5341 aaagacagaa ctcaagcact caaagacaga gctgaacttc aaagagtggc aaaaggcctt
     5401 cacagatgtg atgggaatgg atgaactcta caagacgtaa atgcaaaatc ctttcaagca
     5461 ttcccttctt ctctatcact cttctttctt tttgtcaaaa aattctctcg ctaatttatt
     5521 tgctttttta atgttattat tttatgactt tttatagtca ctgaaaagtt tgcatctgag
     5581 tgaagtgaat gctatcaaaa tgtgattctg tctgatgtac tttcacaatc tctcttcaat
     5641 tccattttga agtgctttaa acccgaaagg ttgagaaaaa tgcgagcgct caaatatttg
     5701 tattgtgttc gttgagtgac ccaacaaaaa gaggaaactt tattgtgccg ccaagaaaaa
     5761 agt
//
