LOCUS       sequence-393489-        6579 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  hygR selection mScarlet 2X  marked rapid RMCE integration vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6579)
  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 6579)
  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: pHygRR2
FEATURES             Location/Qualifiers
     source          1..6579
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    380..404
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(405..1430)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(405..1430)
                     /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(1433..1457)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1936..1953)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2107..2695)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2187..2206)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2866..3657,3658..3726))
                     /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     3489..3508
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3727..3831)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3857..4312)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3994..4015)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4206..4225
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    complement(4324..4357)
                     /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(4427..4460)
                     /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    4481..4514
                     /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             5334..5390
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     terminator      6252..6575
                     /gene="tbb-2"
                     /label=tbb-2 terminator
                     /note="transcription terminator for the C. elegans tubulin 
                     beta-2 gene"
ORIGIN
        1 ctaggcccag ttgtcctcga acaggtctaa aaattgcggt cataaactga aacgtaacat
       61 atgataaggt attttgtgtg cggttttttc tatgatgttt caaaaaaatt tatcagaagt
      121 aaaaaacata agaagtgtac acagaacatt gtgtagattt tgggatagaa ttattattaa
      181 tggggggagg gagcacaatt tttttcgact ttttattacg aattaattct atttttgaat
      241 ctacacaatt tcattgttag aggtgactta aaagaagcta aaaaacaaag aaattaagag
      301 aagttttttg ataataacaa aaataggggg tgggagcaca gggagaaaga gcatgtaggg
      361 atgttgaaga gtaattggac ccactttgta caagaaagct gggtctattc ctttgccctc
      421 ggacgagtgc tggggcgtcg gtttccacta tcggcgagta cttctacaca gccatcggtc
      481 cagacggccg cgcttctgcg ggcgatttgt gtacgcccga cagtcccggc tccggatcgg
      541 acgattgcgt cgcatcgacc ctgcgcccaa gctgcatcat cgaaattgcc gtcaaccaag
      601 ctctgataga gttggtcaag accaatgcgg agcatatacg cccggagccg cggcgatcct
      661 gcaagctccg gatgcctccg ctcgaagtag cgcgtctgct gctccataca agccaaccac
      721 ggcctccaga agaagatgtt ggcgacctcg tattgggaat ccccgaacat cgcctcgctc
      781 cagtcaatga ccgctgttat gcggccattg tccgtcagga cattgttgga gccgaaatcc
      841 gcgtgcacga ggtgccggac ttcggggcag tcctcggccc aaagcatcag ctcatcgaga
      901 gcctgcgcga cggacgcact gacggtgtcg tccatcacag tttgccagtg atacacatgg
      961 ggatcagcaa tcgcgcatat gaaatcacgc catgtagtgt attgaccgat tccttgcggt
     1021 ccgaatgggc cgaacccgct cgtctggcta agatcggccg cagcgatcgc atccatggcc
     1081 tccgcgaccg gctgcagaac agcgggcagt tcggtttcag gcaggtcttg caacgtgaca
     1141 ccctgtgcac ggcgggagat gcaataggtc aggctctcgc tgaattcccc aatgtcaagc
     1201 acttccggaa tcgggagcgc ggccgatgca aagtgccgat aaacataacg atctttgtag
     1261 aaaccatcgg cgcagctatt tacccgcagg acatatccac gccctcctac atcgaagctg
     1321 aaagcacgag attcttcgcc ctccgagagc tgcatcaggt cggagacgct gtcgaacttt
     1381 tcgatcagaa acttctcgac agacgtcgcg gtgagttcag gctttttcat ggagcctgct
     1441 tttttgtaca aacttgcggc accgcctgac atattacctt aaaattcaaa aattaatttc
     1501 agatcagtca taaccaagaa aacaaaataa acgttaggac agaaaattca aattaattaa
     1561 aaattatgct gaataagaaa attacatgca aagccttatt ttcctcatag aaacagttga
     1621 ataacatatt ttttaattga aaacatagca aaattcgtgg taaaatttgt caaaaccttg
     1681 gaaaaaatcg aaaaaagaac atttcccgat gcattttcct atttctcgtt gccgacaacg
     1741 tataaacgtt gaatactcag tcaacgaatt tggctgctgg cttggactct cgcacggaag
     1801 agacgcgtgt gtagatttac gacgaaagca aaaatgatcc cgctttccag tcgggaaacc
     1861 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     1921 ggcgaattcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     1981 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     2041 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     2101 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     2161 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     2221 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     2281 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     2341 tcgctccaag ctgggctgtg tgcacgaacc ccccattcag cccgaccgct gcgccttatc
     2401 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     2461 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     2521 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     2581 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     2641 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     2701 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     2761 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     2821 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     2881 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     2941 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     3001 accgcgagat ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     3061 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     3121 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     3181 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     3241 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     3301 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     3361 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     3421 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     3481 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     3541 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     3601 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     3661 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     3721 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     3781 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     3841 ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     3901 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     3961 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     4021 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     4081 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     4141 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     4201 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     4261 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcgtcggag
     4321 ctcataactt cgtatagcat acattatacg aagttatgcg gccgctctag aactagtggc
     4381 gaagagcctg caggtaacca cgcgttgcat gctcttcggt acccgggaag ttcctatact
     4441 atttgaagaa taggaacttc tgcaggaatt cgatatcggt gaagttccta ttctctagaa
     4501 agtataggaa cttcaaaatg ggatctatgg taagtaaagg agaggctgtc attaaagagt
     4561 tcatgcgttt caaggtaagt tttcttgaga cctttactaa ctaactaggt ctcatagatt
     4621 ttcaggttca catggaagga tctatgaacg gacacgaatt cgaaatcgaa ggagaaggag
     4681 aaggacgccc atacgaggga actcaaactg ctaagcttaa ggttactaaa ggaggaccac
     4741 ttccattctc ttgggatatc ctttctccac agttcatgta cggatctcgc gctttcacca
     4801 agcacccagc tgatatccca gattactaca agcagtcttt cccagaagga ttcaaatggg
     4861 agcgcgttat gaacttcgaa gatggtggag ctgttaccgt tacccaagat acctcccttg
     4921 aggatggaac ccttatctac aaggtaagtt atgtagagac cttaatactc aactatggtc
     4981 tcaaacattt tcaggttaag cttcgcggaa ctaatttccc accagatgga ccagttatgc
     5041 agaagaagac aatgggatgg gaagcttcta ccgagcgcct ttacccagag gatggagtcc
     5101 ttaagggaga tattaagatg gctcttagac ttaaggatgg aggacgttac cttgctgatt
     5161 tcaagactac ttacaaggct aagaagccag tgcagatgcc aggagcttac aacgttgatc
     5221 gtaagcttga tatcacctct cacaacgagg attacaccgt tgtcgagcaa tacgagcgct
     5281 cagagggacg ccactctacc ggaggaatgg atgagcttta caagggatcc ggagctacca
     5341 acttctccct tctcaagcag gctggagacg ttgaggagaa cccaggaccc atgggtatgg
     5401 tttccaaggg agaggccgtc atcaaggagt tcatgcgttt caaggtccac atggagggat
     5461 ccatgaacgg acacgagttc gagatcgagg gagagggaga gggacgtcca tacgagggaa
     5521 cccaaaccgc caagctcaag gtcaccaagg taagtttaaa catatatata ctaactaacc
     5581 ctgattattt aaattttcag ggaggaccac tcccattctc ctgggacatc ctctccccac
     5641 aattcatgta cggatcccgt gccttcacca agcacccagc cgacatccca gactactaca
     5701 agcaatcctt cccagaggga ttcaagtggg agcgtgtcat gaacttcgag gacggaggag
     5761 ccgtcaccgt cacccaagac acctccctcg aggacggaac cctcatctac aaggtaagtt
     5821 taaacagttc ggtactaact aaccatacat atttaaattt tcaggtcaag ctccgtggaa
     5881 ccaacttccc accagacgga ccagtcatgc aaaagaagac catgggatgg gaggcctcca
     5941 ccgagcgtct ctacccagag gacggagtcc tcaagggaga catcaagatg gccctccgtc
     6001 tcaaggacgg aggacgttac ctcgccgact tcaaggtaag tttaaacatg attttactaa
     6061 ctaactaatc tgatttaaat tttcagacca cctacaaggc caagaagcca gtccaaatgc
     6121 caggagccta caacgtcgac cgtaagctcg acatcacctc ccacaacgag gactacaccg
     6181 tcgtcgagca atacgagcgt tccgagggac gtcactccac cggaggaatg gacgagctct
     6241 acaagacgta aatgcaaaat cctttcaagc attcccttct tctctatcac tcttctttct
     6301 ttttgtcaaa aaattctctc gctaatttat ttgctttttt aatgttatta ttttatgact
     6361 ttttatagtc actgaaaagt ttgcatctga gtgaagtgaa tgctatcaaa atgtgattct
     6421 gtctgatgta ctttcacaat ctctcttcaa ttccattttg aagtgcttta aacccgaaag
     6481 gttgagaaaa atgcgagcgc tcaaatattt gtattgtgtt cgttgagtga cccaacaaaa
     6541 agaggaaact ttattgtgcc gccaagaaaa aagtctcac
//
