LOCUS       sequence-485494-        6562 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 6562)
  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 6562)
  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: pHygGP6
FEATURES             Location/Qualifiers
     source          1..6562
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    90..123
                     /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             136..156
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    1970..1994
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(1995..3020)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(1995..3020)
                     /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(3023..3047)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(3526..3543)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3697..4285)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3777..3796)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4456..5247,5248..5316))
                     /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     5079..5098
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5317..5421)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5447..5902)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5584..5605)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5796..5815
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    complement(5914..5947)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    6064..6163
                     /label=phage phi-C31 attP
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attachment site of phage phi-C31"
ORIGIN
        1 gtacggcgac gtaacgtgag ataggcggaa cccgggaagt tcctatacta tttgaagaat
       61 aggaacttct gcaggaattc gatatcggtg aagttcctat tctctagaaa gtataggaac
      121 ttcaaaatgg gatccccaaa gaagaagcgt aaggtgtcga tgagtaaagg agaagaattg
      181 ttcactggag ttgtcccaat cctcgtcgag ctcgacggag acgtcaacgg acacaagttc
      241 tccgtctccg gagagggaga gggagacgcc acctacggaa agctcaccct caagttcatc
      301 tgcaccaccg gaaagctccc agtcccatgg ccaaccctcg tcaccacctt ctgctacgga
      361 gtccaatgct tctcccgtta cccagaccac atgaagcgtc acgacttctt caagtccgcc
      421 atgccagagg gatacgtcca agagcgtacc atcttcttca aggtaagttt aaacatatat
      481 atactaacta ctgattattt aaattttcag gacgacggaa actacaagac ccgtgccgag
      541 gtcaagttcg agggagacac cctcgtcaac cgtatcgagc tcaaggtaag tttaaacagt
      601 tcggtactaa ctaaccatac atatttaaat tttcagggaa tcgacttcaa ggaggacgga
      661 aacatcctcg gacacaagct cgagtacaac tacaactccc acaacgtcta catcatggcc
      721 gacaagcaaa agaacggaat caaggtcaac ttcaaggtaa gtttaaacat gattttacta
      781 actaactaat ctgatttaaa ttttcagatc cgtcacaaca tcgaggacgg atccgtccaa
      841 ctcgccgacc actaccaaca aaacacccca atcggagacg gaccagtcct cctcccagac
      901 aaccactacc tctccaccca atccgccctc tccaaggacc caaacgagaa gcgtgaccac
      961 atggtcctcc tcgagttcgt caccgccgcc ggaatcaccc acggaatgga cgagctctac
     1021 aaggcctccc gtcgtcgtaa ggccaaccca accaagctct ccgagaacgc caagaagctc
     1081 gccaagtaga tcaactgctc taattccatc tcgtttctct cttttctttc aatctcttta
     1141 cccgtttctc ttcccctacg tcccccaaac ctaccactgc acagtaacac atacacattc
     1201 aacgtgtcac aataaaagcc catcaacacc ttctctgttg ttttttttct tcaaatgcat
     1261 ttttttcact ttgtttaata atttttacgc atcggatttg taggatagtg gaaatttgtc
     1321 cttcaatgct tcaattgatt cgacacttct caatattttt gatcaataaa ccattgattt
     1381 tctatttaga agaagatcac tgtaaataac accaaaaaca tcacagcaaa aaaactccca
     1441 ctcaattata ttactgtttt tattgaaact agaataaatt tgaaaatctt caaagcatac
     1501 acaattaaaa aaaacgaaaa aagaaaaagt cccgttgatt ctcaaactta aatttttaaa
     1561 cttgtctgaa ccatgttgta agtttgtaaa agtttcccag ttgtcctcga acaggtctaa
     1621 aaattgcggt cataaactga aacgtaacat atgataaggt attttgtgtg cggttttttc
     1681 tatgatgttt caaaaaaatt tatcagaagt aaaaaacata agaagtgtac acagaacatt
     1741 gtgtagattt tgggatagaa ttattattaa tggggggagg gagcacaatt tttttcgact
     1801 ttttattacg aattaattct atttttgaat ctacacaatt tcattgttag aggtgactta
     1861 aaagaagcta aaaaacaaag aaattaagag aagttttttg ataataacaa aaataggggg
     1921 tgggagcaca gggagaaaga gcatgtaggg atgttgaaga gtaattggac ccactttgta
     1981 caagaaagct gggtctattc ctttgccctc ggacgagtgc tggggcgtcg gtttccacta
     2041 tcggcgagta cttctacaca gccatcggtc cagacggccg cgcttctgcg ggcgatttgt
     2101 gtacgcccga cagtcccggc tccggatcgg acgattgcgt cgcatcgacc ctgcgcccaa
     2161 gctgcatcat cgaaattgcc gtcaaccaag ctctgataga gttggtcaag accaatgcgg
     2221 agcatatacg cccggagccg cggcgatcct gcaagctccg gatgcctccg ctcgaagtag
     2281 cgcgtctgct gctccataca agccaaccac ggcctccaga agaagatgtt ggcgacctcg
     2341 tattgggaat ccccgaacat cgcctcgctc cagtcaatga ccgctgttat gcggccattg
     2401 tccgtcagga cattgttgga gccgaaatcc gcgtgcacga ggtgccggac ttcggggcag
     2461 tcctcggccc aaagcatcag ctcatcgaga gcctgcgcga cggacgcact gacggtgtcg
     2521 tccatcacag tttgccagtg atacacatgg ggatcagcaa tcgcgcatat gaaatcacgc
     2581 catgtagtgt attgaccgat tccttgcggt ccgaatgggc cgaacccgct cgtctggcta
     2641 agatcggccg cagcgatcgc atccatggcc tccgcgaccg gctgcagaac agcgggcagt
     2701 tcggtttcag gcaggtcttg caacgtgaca ccctgtgcac ggcgggagat gcaataggtc
     2761 aggctctcgc tgaattcccc aatgtcaagc acttccggaa tcgggagcgc ggccgatgca
     2821 aagtgccgat aaacataacg atctttgtag aaaccatcgg cgcagctatt tacccgcagg
     2881 acatatccac gccctcctac atcgaagctg aaagcacgag attcttcgcc ctccgagagc
     2941 tgcatcaggt cggagacgct gtcgaacttt tcgatcagaa acttctcgac agacgtcgcg
     3001 gtgagttcag gctttttcat ggagcctgct tttttgtaca aacttgcggc accgcctgac
     3061 atattacctt aaaattcaaa aattaatttc agatcagtca taaccaagaa aacaaaataa
     3121 acgttaggac agaaaattca aattaattaa aaattatgct gaataagaaa attacatgca
     3181 aagccttatt ttcctcatag aaacagttga ataacatatt ttttaattga aaacatagca
     3241 aaattcgtgg taaaatttgt caaaaccttg gaaaaaatcg aaaaaagaac atttcccgat
     3301 gcattttcct atttctcgtt gccgacaacg tataaacgtt gaatactcag tcaacgaatt
     3361 tggctgctgg cttggactct cgcacggaag agacgcgtgt gtagatttac gacgaaagca
     3421 aaaatgatcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     3481 aacgcgcggg gagaggcggt ttgcgtattg ggcgaattcc gcttcctcgc tcactgactc
     3541 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     3601 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     3661 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     3721 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     3781 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     3841 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     3901 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     3961 ccccattcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     4021 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     4081 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     4141 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     4201 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     4261 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     4321 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     4381 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     4441 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     4501 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     4561 cttaccatct ggccccagtg ctgcaatgat accgcgagat ccacgctcac cggctccaga
     4621 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     4681 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     4741 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     4801 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     4861 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     4921 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     4981 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     5041 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     5101 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     5161 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     5221 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     5281 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     5341 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     5401 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt
     5461 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     5521 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     5581 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     5641 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     5701 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     5761 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     5821 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     5881 aatgcgccgc tacagggcgc gtcgtcggag ctcataactt cgtatagcat acattatacg
     5941 aagttatgcg gccgctctag aactagtggc tgacatccag tagacggagg ctaccggtgt
     6001 actgacggac acaccgaagc cccggcggca accctcagcg gatgccccgg ggcttcacgt
     6061 tttcccaggt cagaagcggt tttcgggagt agtgccccaa ctggggtaac ctttgagttc
     6121 tctcagttgg gggcgtaggg tcgccgacat gacacaaggg gttgtgaccg gggtggacac
     6181 gtacgcgggt gcttacgacc gtcagtcgcg cgagcgcgac aattgtctaa acattttttg
     6241 ttgacttaca ataaattgtt gatataattt attgtgaaat gggtattctt gaatgatttg
     6301 attttaacga agagaacgct gaaatcaact gaacacctcg aattgagatt ttggggattt
     6361 tataccaagt tatcgacgac gcaaatggtg caaactttat attttcaaat attctggaag
     6421 cattcaatgg tcttttaaga aacggacaac tccattcttg atatgcaatg ttttttaaaa
     6481 gaaatctctt taatcgagac gggcgcatat agaacctagg tggcgaagag cctgcaggta
     6541 accacgcgtt gcatgctctt cc
//
