LOCUS       sequence-485485-        6549 bp    DNA     circular SYN 08-SEP-2025
DEFINITION  hygR selection nls-Scarlet marked rapid RMCE integration vector with
            an attP site for RMHE.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6549)
  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 6549)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 8, 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: pHygRP12
FEATURES             Location/Qualifiers
     source          1..6549
                     /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    2638..2737
                     /label=phage phi-C31 attP
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attachment site of phage phi-C31"
     protein_bind    3228..3261
                     /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             3274..3294
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    5093..5117
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(5118..6143)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(5118..6143)
                     /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(6146..6170)
                     /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 gaggctaccg gtgtactgac
     2581 ggacacaccg aagccccggc ggcaaccctc agcggatgcc ccggggcttc acgttttccc
     2641 aggtcagaag cggttttcgg gagtagtgcc ccaactgggg taacctttga gttctctcag
     2701 ttgggggcgt agggtcgccg acatgacaca aggggttgtg accggggtgg acacgtacgc
     2761 gggtgcttac gaccgtcagt cgcgcgagcg cgacaattgt ctaaacattt tttgttgact
     2821 tacaataaat tgttgatata atttattgtg aaatgggtat tcttgaatga tttgatttta
     2881 acgaagagaa cgctgaaatc aactgaacac ctcgaattga gattttgggg attttatacc
     2941 aagttatcga cgacgcaaat ggtgcaaact ttatattttc aaatattctg gaagcattca
     3001 atggtctttt aagaaacgga caactccatt cttgatatgc aatgtttttt aaaagaaatc
     3061 tctttaatcg agacgggcgc atatagaacc tagtggaacc gaagagcctg caggtaacca
     3121 cgcgttgcat gctcttccgt acggcgacgt aacgtgagat aggcggaacc cgggaagttc
     3181 ctatactatt tgaagaatag gaacttctgc aggaattcga tatcggtgaa gttcctattc
     3241 tctagaaagt ataggaactt caaaatggga tccccaaaga agaagcgtaa ggtgtcgatg
     3301 gtttccaagg gagaggccgt catcaaggag ttcatgcgtt tcaaggtcca catggaggga
     3361 tccatgaacg gacacgagtt cgagatcgag ggagagggag agggacgtcc atacgaggga
     3421 acccaaaccg ccaagctcaa ggtcaccaag gtaagtttaa acatatatat actaactaac
     3481 cctgattatt taaattttca gggaggacca ctcccattct cctgggacat cctctcccca
     3541 caattcatgt acggatcccg tgccttcacc aagcacccag ccgacatccc agactactac
     3601 aagcaatcct tcccagaggg attcaagtgg gagcgtgtca tgaacttcga ggacggagga
     3661 gccgtcaccg tcacccaaga cacctccctc gaggacggaa ccctcatcta caaggtaagt
     3721 ttaaacagtt cggtactaac taaccataca tatttaaatt ttcaggtcaa gctccgtgga
     3781 accaacttcc caccagacgg accagtcatg caaaagaaga ccatgggatg ggaggcctcc
     3841 accgagcgtc tctacccaga ggacggagtc ctcaagggag acatcaagat ggccctccgt
     3901 ctcaaggacg gaggacgtta cctcgccgac ttcaaggtaa gtttaaacat gattttacta
     3961 actaactaat ctgatttaaa ttttcagacc acctacaagg ccaagaagcc agtccaaatg
     4021 ccaggagcct acaacgtcga ccgtaagctc gacatcacct cccacaacga ggactacacc
     4081 gtcgtcgagc aatacgagcg ttccgaggga cgtcactcca ccggaggaat ggacgagctc
     4141 tacaaggcct cccgtcgtcg taaggccaac ccaaccaagc tctccgagaa cgccaagaag
     4201 ctcgccaagt agatcaactg ctctaattcc atctcgtttc tctcttttct ttcaatctct
     4261 ttacccgttt ctcttcccct acgtccccca aacctaccac tgcacagtaa cacatacaca
     4321 ttcaacgtgt cacaataaaa gcccatcaac accttctctg ttgttttttt tcttcaaatg
     4381 catttttttc actttgttta ataattttta cgcatcggat ttgtaggata gtggaaattt
     4441 gtccttcaat gcttcaattg attcgacact tctcaatatt tttgatcaat aaaccattga
     4501 ttttctattt agaagaagat cactgtaaat aacaccaaaa acatcacagc aaaaaaactc
     4561 ccactcaatt atattactgt ttttattgaa actagaataa atttgaaaat cttcaaagca
     4621 tacacaatta aaaaaaacga aaaaagaaaa agtcccgttg attctcaaac ttaaattttt
     4681 aaacttgtct gaaccatgtt gtaagtttgt aaaagtttcc cagttgtcct cgaacaggtc
     4741 taaaaattgc ggtcataaac tgaaacgtaa catatgataa ggtattttgt gtgcggtttt
     4801 ttctatgatg tttcaaaaaa atttatcaga agtaaaaaac ataagaagtg tacacagaac
     4861 attgtgtaga ttttgggata gaattattat taatgggggg agggagcaca atttttttcg
     4921 actttttatt acgaattaat tctatttttg aatctacaca atttcattgt tagaggtgac
     4981 ttaaaagaag ctaaaaaaca aagaaattaa gagaagtttt ttgataataa caaaaatagg
     5041 gggtgggagc acagggagaa agagcatgta gggatgttga agagtaattg gacccacttt
     5101 gtacaagaaa gctgggtcta ttcctttgcc ctcggacgag tgctggggcg tcggtttcca
     5161 ctatcggcga gtacttctac acagccatcg gtccagacgg ccgcgcttct gcgggcgatt
     5221 tgtgtacgcc cgacagtccc ggctccggat cggacgattg cgtcgcatcg accctgcgcc
     5281 caagctgcat catcgaaatt gccgtcaacc aagctctgat agagttggtc aagaccaatg
     5341 cggagcatat acgcccggag ccgcggcgat cctgcaagct ccggatgcct ccgctcgaag
     5401 tagcgcgtct gctgctccat acaagccaac cacggcctcc agaagaagat gttggcgacc
     5461 tcgtattggg aatccccgaa catcgcctcg ctccagtcaa tgaccgctgt tatgcggcca
     5521 ttgtccgtca ggacattgtt ggagccgaaa tccgcgtgca cgaggtgccg gacttcgggg
     5581 cagtcctcgg cccaaagcat cagctcatcg agagcctgcg cgacggacgc actgacggtg
     5641 tcgtccatca cagtttgcca gtgatacaca tggggatcag caatcgcgca tatgaaatca
     5701 cgccatgtag tgtattgacc gattccttgc ggtccgaatg ggccgaaccc gctcgtctgg
     5761 ctaagatcgg ccgcagcgat cgcatccatg gcctccgcga ccggctgcag aacagcgggc
     5821 agttcggttt caggcaggtc ttgcaacgtg acaccctgtg cacggcggga gatgcaatag
     5881 gtcaggctct cgctgaattc cccaatgtca agcacttccg gaatcgggag cgcggccgat
     5941 gcaaagtgcc gataaacata acgatctttg tagaaaccat cggcgcagct atttacccgc
     6001 aggacatatc cacgccctcc tacatcgaag ctgaaagcac gagattcttc gccctccgag
     6061 agctgcatca ggtcggagac gctgtcgaac ttttcgatca gaaacttctc gacagacgtc
     6121 gcggtgagtt caggcttttt catggagcct gcttttttgt acaaacttgc ggcaccgcct
     6181 gacatattac cttaaaattc aaaaattaat ttcagatcag tcataaccaa gaaaacaaaa
     6241 taaacgttag gacagaaaat tcaaattaat taaaaattat gctgaataag aaaattacat
     6301 gcaaagcctt attttcctca tagaaacagt tgaataacat attttttaat tgaaaacata
     6361 gcaaaattcg tggtaaaatt tgtcaaaacc ttggaaaaaa tcgaaaaaag aacatttccc
     6421 gatgcatttt cctatttctc gttgccgaca acgtataaac gttgaatact cagtcaacga
     6481 atttggctgc tggcttggac tctcgcacgg aagagacgcg tgtgtagatt tacgacgaaa
     6541 gcaaaaatg
//
