LOCUS       sequence-485995-        6579 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  hygR selection nls-Scarlet marked rapid RMCE integration vector with
            an attB site for RMHE.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6579)
  AUTHORS   Nonet ML
  TITLE     Creation and manipulation of bipartite expression transgenes in C. 
            elegans using phiC31 recombinase.
  JOURNAL   bioRxiv 2024.03.01.583017
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: "bioRxiv 
            2024.03.01.583017"
COMMENT     Sequence Label: pHygRB12
FEATURES             Location/Qualifiers
     source          1..6579
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(100..117)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(271..859)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(351..370)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1030..1821,1822..1890))
                     /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     1653..1672
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1891..1995)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2021..2476)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2158..2179)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2370..2389
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    complement(2488..2521)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    2903..2972
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     protein_bind    complement(3204..3237)
                     /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    3258..3291
                     /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             3304..3324
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    5123..5147
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(5148..6173)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(5148..6173)
                     /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(6176..6200)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 atcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
       61 cggggagagg cggtttgcgt attgggcgaa ttccgcttcc tcgctcactg actcgctgcg
      121 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
      181 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
      241 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
      301 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
      361 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
      421 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
      481 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccat
      541 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
      601 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
      661 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
      721 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
      781 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
      841 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
      901 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
      961 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     1021 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     1081 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     1141 atctggcccc agtgctgcaa tgataccgcg agatccacgc tcaccggctc cagatttatc
     1201 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     1261 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     1321 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     1381 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     1441 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     1501 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     1561 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     1621 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     1681 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     1741 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     1801 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     1861 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     1921 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     1981 aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt
     2041 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     2101 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     2161 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     2221 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     2281 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     2341 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     2401 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     2461 ccgctacagg gcgcgtcgtc ggagctcata acttcgtata gcatacatta tacgaagtta
     2521 tgcggccgct ctagaactag tggctgacat ccagtagacg gaggctcaat tgaaagtaaa
     2581 tattccctgt gtttaattgt tattgttgtg gtggataata gatatttaat ctgccaaaat
     2641 gtgttctttt tacatagctg atcaatttcc agatatatcg agacttgatg tcattcctgg
     2701 atgaatcgtc taatggtgtt aaacctgatt tgattagttg tctctttgtc gtattagagt
     2761 ggatgagttc atccagttgt tgaaaaaatg ttcttattgt gaagaaaaac taattcgtag
     2821 aacctagtgg aaccgaagag cctgcaggta accacgcgtt gcatgctctt ccgtacgtac
     2881 cgtcgacgat gtaggtcacg gtctcgaagc cgcggtgcgg gtgccagggc gtgcccttgg
     2941 gctccccggg cgcgtactcc acctcaccca tctggtccat catgatgaac gggtcgaggt
     3001 ggcggtagtt gatcccggcg aacgcgcggc gcaccgggaa gccctcgccc tcgaaaccgc
     3061 tgggcgcggt ggtcacggtg agcacgggac gtgcgacggc gtcggcgggt gcggatacgc
     3121 ggggcagcgt cagcgggttc tcgacggtca cggcgggcat gtcgacactt aagcgacgta
     3181 acgtgagata ggcggaacac cgggaagttc ctatactatt tgaagaatag gaacttctgc
     3241 aggaattcga tatcggtgaa gttcctattc tctagaaagt ataggaactt caaaatggga
     3301 tccccaaaga agaagcgtaa ggtgtcgatg gtttccaagg gagaggccgt catcaaggag
     3361 ttcatgcgtt tcaaggtcca catggaggga tccatgaacg gacacgagtt cgagatcgag
     3421 ggagagggag agggacgtcc atacgaggga acccaaaccg ccaagctcaa ggtcaccaag
     3481 gtaagtttaa acatatatat actaactaac cctgattatt taaattttca gggaggacca
     3541 ctcccattct cctgggacat cctctcccca caattcatgt acggatcccg tgccttcacc
     3601 aagcacccag ccgacatccc agactactac aagcaatcct tcccagaggg attcaagtgg
     3661 gagcgtgtca tgaacttcga ggacggagga gccgtcaccg tcacccaaga cacctccctc
     3721 gaggacggaa ccctcatcta caaggtaagt ttaaacagtt cggtactaac taaccataca
     3781 tatttaaatt ttcaggtcaa gctccgtgga accaacttcc caccagacgg accagtcatg
     3841 caaaagaaga ccatgggatg ggaggcctcc accgagcgtc tctacccaga ggacggagtc
     3901 ctcaagggag acatcaagat ggccctccgt ctcaaggacg gaggacgtta cctcgccgac
     3961 ttcaaggtaa gtttaaacat gattttacta actaactaat ctgatttaaa ttttcagacc
     4021 acctacaagg ccaagaagcc agtccaaatg ccaggagcct acaacgtcga ccgtaagctc
     4081 gacatcacct cccacaacga ggactacacc gtcgtcgagc aatacgagcg ttccgaggga
     4141 cgtcactcca ccggaggaat ggacgagctc tacaaggcct cccgtcgtcg taaggccaac
     4201 ccaaccaagc tctccgagaa cgccaagaag ctcgccaagt agatcaactg ctctaattcc
     4261 atctcgtttc tctcttttct ttcaatctct ttacccgttt ctcttcccct acgtccccca
     4321 aacctaccac tgcacagtaa cacatacaca ttcaacgtgt cacaataaaa gcccatcaac
     4381 accttctctg ttgttttttt tcttcaaatg catttttttc actttgttta ataattttta
     4441 cgcatcggat ttgtaggata gtggaaattt gtccttcaat gcttcaattg attcgacact
     4501 tctcaatatt tttgatcaat aaaccattga ttttctattt agaagaagat cactgtaaat
     4561 aacaccaaaa acatcacagc aaaaaaactc ccactcaatt atattactgt ttttattgaa
     4621 actagaataa atttgaaaat cttcaaagca tacacaatta aaaaaaacga aaaaagaaaa
     4681 agtcccgttg attctcaaac ttaaattttt aaacttgtct gaaccatgtt gtaagtttgt
     4741 aaaagtttcc cagttgtcct cgaacaggtc taaaaattgc ggtcataaac tgaaacgtaa
     4801 catatgataa ggtattttgt gtgcggtttt ttctatgatg tttcaaaaaa atttatcaga
     4861 agtaaaaaac ataagaagtg tacacagaac attgtgtaga ttttgggata gaattattat
     4921 taatgggggg agggagcaca atttttttcg actttttatt acgaattaat tctatttttg
     4981 aatctacaca atttcattgt tagaggtgac ttaaaagaag ctaaaaaaca aagaaattaa
     5041 gagaagtttt ttgataataa caaaaatagg gggtgggagc acagggagaa agagcatgta
     5101 gggatgttga agagtaattg gacccacttt gtacaagaaa gctgggtcta ttcctttgcc
     5161 ctcggacgag tgctggggcg tcggtttcca ctatcggcga gtacttctac acagccatcg
     5221 gtccagacgg ccgcgcttct gcgggcgatt tgtgtacgcc cgacagtccc ggctccggat
     5281 cggacgattg cgtcgcatcg accctgcgcc caagctgcat catcgaaatt gccgtcaacc
     5341 aagctctgat agagttggtc aagaccaatg cggagcatat acgcccggag ccgcggcgat
     5401 cctgcaagct ccggatgcct ccgctcgaag tagcgcgtct gctgctccat acaagccaac
     5461 cacggcctcc agaagaagat gttggcgacc tcgtattggg aatccccgaa catcgcctcg
     5521 ctccagtcaa tgaccgctgt tatgcggcca ttgtccgtca ggacattgtt ggagccgaaa
     5581 tccgcgtgca cgaggtgccg gacttcgggg cagtcctcgg cccaaagcat cagctcatcg
     5641 agagcctgcg cgacggacgc actgacggtg tcgtccatca cagtttgcca gtgatacaca
     5701 tggggatcag caatcgcgca tatgaaatca cgccatgtag tgtattgacc gattccttgc
     5761 ggtccgaatg ggccgaaccc gctcgtctgg ctaagatcgg ccgcagcgat cgcatccatg
     5821 gcctccgcga ccggctgcag aacagcgggc agttcggttt caggcaggtc ttgcaacgtg
     5881 acaccctgtg cacggcggga gatgcaatag gtcaggctct cgctgaattc cccaatgtca
     5941 agcacttccg gaatcgggag cgcggccgat gcaaagtgcc gataaacata acgatctttg
     6001 tagaaaccat cggcgcagct atttacccgc aggacatatc cacgccctcc tacatcgaag
     6061 ctgaaagcac gagattcttc gccctccgag agctgcatca ggtcggagac gctgtcgaac
     6121 ttttcgatca gaaacttctc gacagacgtc gcggtgagtt caggcttttt catggagcct
     6181 gcttttttgt acaaacttgc ggcaccgcct gacatattac cttaaaattc aaaaattaat
     6241 ttcagatcag tcataaccaa gaaaacaaaa taaacgttag gacagaaaat tcaaattaat
     6301 taaaaattat gctgaataag aaaattacat gcaaagcctt attttcctca tagaaacagt
     6361 tgaataacat attttttaat tgaaaacata gcaaaattcg tggtaaaatt tgtcaaaacc
     6421 ttggaaaaaa tcgaaaaaag aacatttccc gatgcatttt cctatttctc gttgccgaca
     6481 acgtataaac gttgaatact cagtcaacga atttggctgc tggcttggac tctcgcacgg
     6541 aagagacgcg tgtgtagatt tacgacgaaa gcaaaaatg
//
