LOCUS       sequence-485493-        6564 bp    DNA     circular SYN 08-SEP-2025
DEFINITION  hygR selection nls-GFP 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 6564)
  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 6564)
  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: pHygGP16
FEATURES             Location/Qualifiers
     source          1..6564
                     /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    5108..5132
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(5133..6158)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(5133..6158)
                     /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(6161..6185)
                     /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 agtaaaggag aagaattgtt cactggagtt gtcccaatcc tcgtcgagct cgacggagac
     3361 gtcaacggac acaagttctc cgtctccgga gagggagagg gagacgccac ctacggaaag
     3421 ctcaccctca agttcatctg caccaccgga aagctcccag tcccatggcc aaccctcgtc
     3481 accaccttct gctacggagt ccaatgcttc tcccgttacc cagaccacat gaagcgtcac
     3541 gacttcttca agtccgccat gccagaggga tacgtccaag agcgtaccat cttcttcaag
     3601 gtaagtttaa acatatatat actaactact gattatttaa attttcagga cgacggaaac
     3661 tacaagaccc gtgccgaggt caagttcgag ggagacaccc tcgtcaaccg tatcgagctc
     3721 aaggtaagtt taaacagttc ggtactaact aaccatacat atttaaattt tcagggaatc
     3781 gacttcaagg aggacggaaa catcctcgga cacaagctcg agtacaacta caactcccac
     3841 aacgtctaca tcatggccga caagcaaaag aacggaatca aggtcaactt caaggtaagt
     3901 ttaaacatga ttttactaac taactaatct gatttaaatt ttcagatccg tcacaacatc
     3961 gaggacggat ccgtccaact cgccgaccac taccaacaaa acaccccaat cggagacgga
     4021 ccagtcctcc tcccagacaa ccactacctc tccacccaat ccgccctctc caaggaccca
     4081 aacgagaagc gtgaccacat ggtcctcctc gagttcgtca ccgccgccgg aatcacccac
     4141 ggaatggacg agctctacaa ggcctcccgt cgtcgtaagg ccaacccaac caagctctcc
     4201 gagaacgcca agaagctcgc caagtagatc aactgctcta attccatctc gtttctctct
     4261 tttctttcaa tctctttacc cgtttctctt cccctacgtc ccccaaacct accactgcac
     4321 agtaacacat acacattcaa cgtgtcacaa taaaagccca tcaacacctt ctctgttgtt
     4381 ttttttcttc aaatgcattt ttttcacttt gtttaataat ttttacgcat cggatttgta
     4441 ggatagtgga aatttgtcct tcaatgcttc aattgattcg acacttctca atatttttga
     4501 tcaataaacc attgattttc tatttagaag aagatcactg taaataacac caaaaacatc
     4561 acagcaaaaa aactcccact caattatatt actgttttta ttgaaactag aataaatttg
     4621 aaaatcttca aagcatacac aattaaaaaa aacgaaaaaa gaaaaagtcc cgttgattct
     4681 caaacttaaa tttttaaact tgtctgaacc atgttgtaag tttgtaaaag tttcccagtt
     4741 gtcctcgaac aggtctaaaa attgcggtca taaactgaaa cgtaacatat gataaggtat
     4801 tttgtgtgcg gttttttcta tgatgtttca aaaaaattta tcagaagtaa aaaacataag
     4861 aagtgtacac agaacattgt gtagattttg ggatagaatt attattaatg gggggaggga
     4921 gcacaatttt tttcgacttt ttattacgaa ttaattctat ttttgaatct acacaatttc
     4981 attgttagag gtgacttaaa agaagctaaa aaacaaagaa attaagagaa gttttttgat
     5041 aataacaaaa atagggggtg ggagcacagg gagaaagagc atgtagggat gttgaagagt
     5101 aattggaccc actttgtaca agaaagctgg gtctattcct ttgccctcgg acgagtgctg
     5161 gggcgtcggt ttccactatc ggcgagtact tctacacagc catcggtcca gacggccgcg
     5221 cttctgcggg cgatttgtgt acgcccgaca gtcccggctc cggatcggac gattgcgtcg
     5281 catcgaccct gcgcccaagc tgcatcatcg aaattgccgt caaccaagct ctgatagagt
     5341 tggtcaagac caatgcggag catatacgcc cggagccgcg gcgatcctgc aagctccgga
     5401 tgcctccgct cgaagtagcg cgtctgctgc tccatacaag ccaaccacgg cctccagaag
     5461 aagatgttgg cgacctcgta ttgggaatcc ccgaacatcg cctcgctcca gtcaatgacc
     5521 gctgttatgc ggccattgtc cgtcaggaca ttgttggagc cgaaatccgc gtgcacgagg
     5581 tgccggactt cggggcagtc ctcggcccaa agcatcagct catcgagagc ctgcgcgacg
     5641 gacgcactga cggtgtcgtc catcacagtt tgccagtgat acacatgggg atcagcaatc
     5701 gcgcatatga aatcacgcca tgtagtgtat tgaccgattc cttgcggtcc gaatgggccg
     5761 aacccgctcg tctggctaag atcggccgca gcgatcgcat ccatggcctc cgcgaccggc
     5821 tgcagaacag cgggcagttc ggtttcaggc aggtcttgca acgtgacacc ctgtgcacgg
     5881 cgggagatgc aataggtcag gctctcgctg aattccccaa tgtcaagcac ttccggaatc
     5941 gggagcgcgg ccgatgcaaa gtgccgataa acataacgat ctttgtagaa accatcggcg
     6001 cagctattta cccgcaggac atatccacgc cctcctacat cgaagctgaa agcacgagat
     6061 tcttcgccct ccgagagctg catcaggtcg gagacgctgt cgaacttttc gatcagaaac
     6121 ttctcgacag acgtcgcggt gagttcaggc tttttcatgg agcctgcttt tttgtacaaa
     6181 cttgcggcac cgcctgacat attaccttaa aattcaaaaa ttaatttcag atcagtcata
     6241 accaagaaaa caaaataaac gttaggacag aaaattcaaa ttaattaaaa attatgctga
     6301 ataagaaaat tacatgcaaa gccttatttt cctcatagaa acagttgaat aacatatttt
     6361 ttaattgaaa acatagcaaa attcgtggta aaatttgtca aaaccttgga aaaaatcgaa
     6421 aaaagaacat ttcccgatgc attttcctat ttctcgttgc cgacaacgta taaacgttga
     6481 atactcagtc aacgaatttg gctgctggct tggactctcg cacggaagag acgcgtgtgt
     6541 agatttacga cgaaagcaaa aatg
//
