LOCUS       sequence-274683-        4252 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  floxed Hygromycin resistant gene with stop codon franking to pA 
            sequeneces between attL1-attR5 on Gateway entry vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4252)
  TITLE     Minoru Ko Lab plasmids
REFERENCE   2  (bases 1 to 4252)
  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"
COMMENT     Sequence Label: pENTR-L1R5-floxHygpA
FEATURES             Location/Qualifiers
     source          1..4252
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(671..704)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             708..1733
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVHRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     primer_bind     complement(2011..2027)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2011..2027)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     polyA_signal    2142..2276
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2179..2198)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2233..2252
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(2280..2313)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    2315..2438
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2493..2512)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2494..2512)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2517..2533)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2517..2533)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2635..2654)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2646..3455
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2713..2732)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3602..4190
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4091..4110
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctta ataacttcgt atagcataca ttatacgaag ttataccatg aaaaagcctg
      721 aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc
      781 tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg
      841 gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgttcatc
      901 ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg gaattcagcg
      961 agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg
     1021 aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg
     1081 ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca
     1141 ctacgtggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg
     1201 tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg
     1261 ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc
     1321 tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt
     1381 cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt atggagcagc
     1441 agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt
     1501 atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg
     1561 atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc gggactgtcg
     1621 ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt gtagaagtac
     1681 tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa tagtcgagaa
     1741 attgatgatc tattaagcaa taaagacgtc cactaaaatg gaagtttttc ctgtcatact
     1801 ttgttaagaa gggtgagaac agagtaccta cattttgaat ggaaggattg gagctacggg
     1861 ggtgggggtg gggtgggatt agataaatgc ctgctcttta ctgaaggctc tttactattg
     1921 ctttatgata atgtttcata gttggatatc ataatttaaa caagcaaaac caaattaagg
     1981 gccagctcat tcctccactc acgatctata gatccactag ttctagataa gtaatgatca
     2041 taatcagcca tatcacatct gtagaggttt tacttgcttt aaaaaacctc ccacacctcc
     2101 ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt
     2161 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     2221 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatcccaa
     2281 taacttcgta tagcatacat tatacgaagt tatacaactt tgtatacaaa agttgaacga
     2341 gaaacgtaaa atgatataaa tatcaatata ttaaattaga ttttgcataa aaaacagact
     2401 acataatact gtaaaacaca acatatccag tcactatgaa tcaactactt agatggtatt
     2461 agtgacctgt actgcagggc ggccgcgata tcccctatag tgagtcgtat tacatggtca
     2521 tagctgtttc ctggcagctc tggcccgtgt ctcaaaatct ctgatgttac attgcacaag
     2581 ataaaaatat atcatcatga acaataaaac tgtctgctta cataaacagt aatacaaggg
     2641 gtgttatgag ccatattcaa cgggaaacgt cgaggccgcg attaaattcc aacatggatg
     2701 ctgatttata tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct
     2761 atcgcttgta tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg
     2821 ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc
     2881 ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga
     2941 tccccggaaa aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg
     3001 ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt
     3061 ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg
     3121 ttgatgcgag tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag
     3181 aaatgcataa acttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac
     3241 ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg
     3301 gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc
     3361 cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat
     3421 tgcagtttca tttgatgctc gatgagtttt tctaatcaga attggttaat tggttgtaac
     3481 actggcagag cattacgctg acttgacggg acggcgcaag ctcatgacca aaatccctta
     3541 acgtgagtta cgcgtcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     3601 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     3661 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     3721 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     3781 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     3841 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     3901 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     3961 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     4021 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     4081 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     4141 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     4201 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg tt
//
