LOCUS       sequence-485985-        6594 bp    DNA     circular SYN 16-SEP-2025
DEFINITION  hygR selection nls-GFP 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 6594)
  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 6594)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 16, 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: pHygGB16
FEATURES             Location/Qualifiers
     source          1..6594
                     /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    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    5138..5162
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(5163..6188)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(5163..6188)
                     /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(6191..6215)
                     /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 agtaaaggag aagaattgtt cactggagtt
     3361 gtcccaatcc tcgtcgagct cgacggagac gtcaacggac acaagttctc cgtctccgga
     3421 gagggagagg gagacgccac ctacggaaag ctcaccctca agttcatctg caccaccgga
     3481 aagctcccag tcccatggcc aaccctcgtc accaccttct gctacggagt ccaatgcttc
     3541 tcccgttacc cagaccacat gaagcgtcac gacttcttca agtccgccat gccagaggga
     3601 tacgtccaag agcgtaccat cttcttcaag gtaagtttaa acatatatat actaactact
     3661 gattatttaa attttcagga cgacggaaac tacaagaccc gtgccgaggt caagttcgag
     3721 ggagacaccc tcgtcaaccg tatcgagctc aaggtaagtt taaacagttc ggtactaact
     3781 aaccatacat atttaaattt tcagggaatc gacttcaagg aggacggaaa catcctcgga
     3841 cacaagctcg agtacaacta caactcccac aacgtctaca tcatggccga caagcaaaag
     3901 aacggaatca aggtcaactt caaggtaagt ttaaacatga ttttactaac taactaatct
     3961 gatttaaatt ttcagatccg tcacaacatc gaggacggat ccgtccaact cgccgaccac
     4021 taccaacaaa acaccccaat cggagacgga ccagtcctcc tcccagacaa ccactacctc
     4081 tccacccaat ccgccctctc caaggaccca aacgagaagc gtgaccacat ggtcctcctc
     4141 gagttcgtca ccgccgccgg aatcacccac ggaatggacg agctctacaa ggcctcccgt
     4201 cgtcgtaagg ccaacccaac caagctctcc gagaacgcca agaagctcgc caagtagatc
     4261 aactgctcta attccatctc gtttctctct tttctttcaa tctctttacc cgtttctctt
     4321 cccctacgtc ccccaaacct accactgcac agtaacacat acacattcaa cgtgtcacaa
     4381 taaaagccca tcaacacctt ctctgttgtt ttttttcttc aaatgcattt ttttcacttt
     4441 gtttaataat ttttacgcat cggatttgta ggatagtgga aatttgtcct tcaatgcttc
     4501 aattgattcg acacttctca atatttttga tcaataaacc attgattttc tatttagaag
     4561 aagatcactg taaataacac caaaaacatc acagcaaaaa aactcccact caattatatt
     4621 actgttttta ttgaaactag aataaatttg aaaatcttca aagcatacac aattaaaaaa
     4681 aacgaaaaaa gaaaaagtcc cgttgattct caaacttaaa tttttaaact tgtctgaacc
     4741 atgttgtaag tttgtaaaag tttcccagtt gtcctcgaac aggtctaaaa attgcggtca
     4801 taaactgaaa cgtaacatat gataaggtat tttgtgtgcg gttttttcta tgatgtttca
     4861 aaaaaattta tcagaagtaa aaaacataag aagtgtacac agaacattgt gtagattttg
     4921 ggatagaatt attattaatg gggggaggga gcacaatttt tttcgacttt ttattacgaa
     4981 ttaattctat ttttgaatct acacaatttc attgttagag gtgacttaaa agaagctaaa
     5041 aaacaaagaa attaagagaa gttttttgat aataacaaaa atagggggtg ggagcacagg
     5101 gagaaagagc atgtagggat gttgaagagt aattggaccc actttgtaca agaaagctgg
     5161 gtctattcct ttgccctcgg acgagtgctg gggcgtcggt ttccactatc ggcgagtact
     5221 tctacacagc catcggtcca gacggccgcg cttctgcggg cgatttgtgt acgcccgaca
     5281 gtcccggctc cggatcggac gattgcgtcg catcgaccct gcgcccaagc tgcatcatcg
     5341 aaattgccgt caaccaagct ctgatagagt tggtcaagac caatgcggag catatacgcc
     5401 cggagccgcg gcgatcctgc aagctccgga tgcctccgct cgaagtagcg cgtctgctgc
     5461 tccatacaag ccaaccacgg cctccagaag aagatgttgg cgacctcgta ttgggaatcc
     5521 ccgaacatcg cctcgctcca gtcaatgacc gctgttatgc ggccattgtc cgtcaggaca
     5581 ttgttggagc cgaaatccgc gtgcacgagg tgccggactt cggggcagtc ctcggcccaa
     5641 agcatcagct catcgagagc ctgcgcgacg gacgcactga cggtgtcgtc catcacagtt
     5701 tgccagtgat acacatgggg atcagcaatc gcgcatatga aatcacgcca tgtagtgtat
     5761 tgaccgattc cttgcggtcc gaatgggccg aacccgctcg tctggctaag atcggccgca
     5821 gcgatcgcat ccatggcctc cgcgaccggc tgcagaacag cgggcagttc ggtttcaggc
     5881 aggtcttgca acgtgacacc ctgtgcacgg cgggagatgc aataggtcag gctctcgctg
     5941 aattccccaa tgtcaagcac ttccggaatc gggagcgcgg ccgatgcaaa gtgccgataa
     6001 acataacgat ctttgtagaa accatcggcg cagctattta cccgcaggac atatccacgc
     6061 cctcctacat cgaagctgaa agcacgagat tcttcgccct ccgagagctg catcaggtcg
     6121 gagacgctgt cgaacttttc gatcagaaac ttctcgacag acgtcgcggt gagttcaggc
     6181 tttttcatgg agcctgcttt tttgtacaaa cttgcggcac cgcctgacat attaccttaa
     6241 aattcaaaaa ttaatttcag atcagtcata accaagaaaa caaaataaac gttaggacag
     6301 aaaattcaaa ttaattaaaa attatgctga ataagaaaat tacatgcaaa gccttatttt
     6361 cctcatagaa acagttgaat aacatatttt ttaattgaaa acatagcaaa attcgtggta
     6421 aaatttgtca aaaccttgga aaaaatcgaa aaaagaacat ttcccgatgc attttcctat
     6481 ttctcgttgc cgacaacgta taaacgttga atactcagtc aacgaatttg gctgctggct
     6541 tggactctcg cacggaagag acgcgtgtgt agatttacga cgaaagcaaa aatg
//
