LOCUS       sequence-393480-        6039 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 6039)
  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 6039)
  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: pHygG2r
FEATURES             Location/Qualifiers
     source          1..6039
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(101..118)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(272..860)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(352..371)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1031..1822,1823..1891))
                     /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     1654..1673
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1892..1996)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2022..2477)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2159..2180)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2371..2390
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    complement(2489..2522)
                     /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(2602..2635)
                     /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    2656..2689
                     /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             2702..2722
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    4584..4608
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(4609..5634)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(4609..5634)
                     /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(5637..5661)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 gatcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
       61 gcggggagag gcggtttgcg tattgggcga attccgcttc ctcgctcact gactcgctgc
      121 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
      181 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
      241 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
      301 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
      361 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
      421 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
      481 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaacccccca
      541 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
      601 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
      661 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
      721 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
      781 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
      841 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
      901 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
      961 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     1021 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     1081 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     1141 catctggccc cagtgctgca atgataccgc gagatccacg ctcaccggct ccagatttat
     1201 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     1261 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     1321 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     1381 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     1441 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     1501 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     1561 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     1621 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     1681 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     1741 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     1801 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     1861 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     1921 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     1981 aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat
     2041 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     2101 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     2161 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     2221 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     2281 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     2341 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     2401 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     2461 gccgctacag ggcgcgtcgt cggagctcat aacttcgtat agcatacatt atacgaagtt
     2521 atgcggccgc tctagaacta gtggtaccga agagcatgca acgcgtggtt acctgcaggc
     2581 tcttcgccac ttaagacccg ggaagttcct atactatttg aagaatagga acttctgcag
     2641 gaattcgata tcggtgaagt tcctattctc tagaaagtat aggaacttca aaatgggatc
     2701 cccaaagaag aagcgtaagg tgtcgatggt gtcgaaggga gaagaggata acatggcttc
     2761 actcccagct acacacgaac tccacatctt cggatcgatc aacggagtgg atttcgatat
     2821 ggtcggacaa ggtaagttta aacatatata tactaactaa ccctgattat ttaaattttc
     2881 aggaactgga aacccaaacg atggatacga ggaactcaac ctcaagtcga caaagggaga
     2941 tctgcaattc tcgccatgga ttctcgtgcc acacatcgga tacggattcc accaatacct
     3001 cccataccca ggtaagttta aactgagttc tactaactaa cgagtaatat ttaaattttc
     3061 agatggaatg tcaccattcc aagctgccat ggtggatgga tcgggatacc aagttcaccg
     3121 aacaatgcaa ttcgaggatg gagcctcgct cacagtgaac taccgataca catacgaggg
     3181 atcgcacatc aaggtaagtt taaacagttc ggtactaact aaccatacat atttaaattt
     3241 tcagggagag gctcaagtta agggaacagg attcccagct gatggaccag tgatgacaaa
     3301 ctcactcaca gctgctgatt ggtgccgatc gaaaaagaca tacccaaatg ataagacaat
     3361 catctcgaca ttcaagtggt cgtacactac tggaaacgga aagcgatacc gatcgacagc
     3421 ccgaacaaca tacacattcg ctaagccaat ggccgccaac tacctcaagg taagtttaaa
     3481 catgatttta ctaactaact aatctgattt aaattttcag aatcaaccaa tgtacgtgtt
     3541 ccgaaagaca gaactcaagc actcaaagac agagctgaac ttcaaagagt ggcaaaaggc
     3601 cttcacagat gtgatgggaa tggatgaact ctacaaggcc tcccgtcgtc gtaaggccaa
     3661 cccaaccaag ctctccgaga acgccaagaa gctcgccaag tagatcaact gctctaattc
     3721 catctcgttt ctctcttttc tttcaatctc tttacccgtt tctcttcccc tacgtccccc
     3781 aaacctacca ctgcacagta acacatacac attcaacgtg tcacaataaa agcccatcaa
     3841 caccttctct gttgtttttt ttcttcaaat gcattttttt cactttgttt aataattttt
     3901 acgcatcgga tttgtaggat agtggaaatt tgtccttcaa tgcttcaatt gattcgacac
     3961 ttctcaatat ttttgatcaa taaaccattg attttctatt tagaagaaga tcactgtaaa
     4021 taacaccaaa aacatcacag caaaaaaact cccactcaat tatattactg tttttattga
     4081 aactagaata aatttgaaaa tcttcaaagc atacacaatt aaaaaaaacg aaaaaagaaa
     4141 aagtcccgtt gattctcaaa cttaaatttt taaacttgtc tgaaccatgt tgtaagtttg
     4201 taaaagtttc ccagttgtcc tcgaacaggt ctaaaaattg cggtcataaa ctgaaacgta
     4261 acatatgata aggtattttg tgtgcggttt tttctatgat gtttcaaaaa aatttatcag
     4321 aagtaaaaaa cataagaagt gtacacagaa cattgtgtag attttgggat agaattatta
     4381 ttaatggggg gagggagcac aatttttttc gactttttat tacgaattaa ttctattttt
     4441 gaatctacac aatttcattg ttagaggtga cttaaaagaa gctaaaaaac aaagaaatta
     4501 agagaagttt tttgataata acaaaaatag ggggtgggag cacagggaga aagagcatgt
     4561 agggatgttg aagagtaatt ggacccactt tgtacaagaa agctgggtct attcctttgc
     4621 cctcggacga gtgctggggc gtcggtttcc actatcggcg agtacttcta cacagccatc
     4681 ggtccagacg gccgcgcttc tgcgggcgat ttgtgtacgc ccgacagtcc cggctccgga
     4741 tcggacgatt gcgtcgcatc gaccctgcgc ccaagctgca tcatcgaaat tgccgtcaac
     4801 caagctctga tagagttggt caagaccaat gcggagcata tacgcccgga gccgcggcga
     4861 tcctgcaagc tccggatgcc tccgctcgaa gtagcgcgtc tgctgctcca tacaagccaa
     4921 ccacggcctc cagaagaaga tgttggcgac ctcgtattgg gaatccccga acatcgcctc
     4981 gctccagtca atgaccgctg ttatgcggcc attgtccgtc aggacattgt tggagccgaa
     5041 atccgcgtgc acgaggtgcc ggacttcggg gcagtcctcg gcccaaagca tcagctcatc
     5101 gagagcctgc gcgacggacg cactgacggt gtcgtccatc acagtttgcc agtgatacac
     5161 atggggatca gcaatcgcgc atatgaaatc acgccatgta gtgtattgac cgattccttg
     5221 cggtccgaat gggccgaacc cgctcgtctg gctaagatcg gccgcagcga tcgcatccat
     5281 ggcctccgcg accggctgca gaacagcggg cagttcggtt tcaggcaggt cttgcaacgt
     5341 gacaccctgt gcacggcggg agatgcaata ggtcaggctc tcgctgaatt ccccaatgtc
     5401 aagcacttcc ggaatcggga gcgcggccga tgcaaagtgc cgataaacat aacgatcttt
     5461 gtagaaacca tcggcgcagc tatttacccg caggacatat ccacgccctc ctacatcgaa
     5521 gctgaaagca cgagattctt cgccctccga gagctgcatc aggtcggaga cgctgtcgaa
     5581 cttttcgatc agaaacttct cgacagacgt cgcggtgagt tcaggctttt tcatggagcc
     5641 tgcttttttg tacaaacttg cggcaccgcc tgacatatta ccttaaaatt caaaaattaa
     5701 tttcagatca gtcataacca agaaaacaaa ataaacgtta ggacagaaaa ttcaaattaa
     5761 ttaaaaatta tgctgaataa gaaaattaca tgcaaagcct tattttcctc atagaaacag
     5821 ttgaataaca tattttttaa ttgaaaacat agcaaaattc gtggtaaaat ttgtcaaaac
     5881 cttggaaaaa atcgaaaaaa gaacatttcc cgatgcattt tcctatttct cgttgccgac
     5941 aacgtataaa cgttgaatac tcagtcaacg aatttggctg ctggcttgga ctctcgcacg
     6001 gaagagacgc gtgtgtagat ttacgacgaa agcaaaaat
//
