LOCUS       sequence-274621-        4629 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Provides pA sequences N-terminal to floxed PGK-neo-pA cassette on 
            Gateway entry vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4629)
  TITLE     Minoru Ko Lab plasmids
REFERENCE   2  (bases 1 to 4629)
  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-R2L3-pA-floxPGKneopA
FEATURES             Location/Qualifiers
     source          1..4629
                     /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    complement(624..747)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     polyA_signal    752..800
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    867..900
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        965..1464
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1000..1023)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1397..1416
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        1476..1523
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             1542..2345
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1605..1624)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2215..2234
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(2377..2394)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2383..2607
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    2664..2697
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(2713..2729)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2713..2729)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    complement(2738..2833)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2870..2889)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2871..2889)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2894..2910)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2894..2910)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3012..3031)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3023..3832
                     /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(3090..3109)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3979..4567
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4468..4487
                     /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 tcgggccctg cagctctaga gctcgaattc tacaggtcac
      601 taataccatc taagtagttg gttcatagtg actgcatatg ttgtgtttta cagtattatg
      661 tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt
      721 ctcgttcaac tttcttgtac aaagtgggga tcggcaataa aaagacagaa taaaacgcac
      781 gggtgttggg tcgtttgttc gtcgacctgc agccaagcta tcgaattcct gcagcccaat
      841 tccgatcata ttcaataacc cttaatataa cttcgtataa tgtatgctat acgaagttat
      901 taggtctgaa gaggagttta cgtccagcca agctagcttg gctgcaggtc gtcgaaattc
      961 taccgggtag gggaggcgct tttcccaagg cagtctggag catgcgcttt agcagccccg
     1021 ctgggcactt ggcgctacac aagtggcctc tggcctcgca cacattccac atccaccggt
     1081 aggcgccaac cggctccgtt ctttggtggc cccttcgcgc caccttctac tcctccccta
     1141 gtcaggaagt tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca aatggaagta
     1201 gcacgtctca ctagtctcgt gcagatggac agcaccgctg agcaatggaa gcgggtaggc
     1261 ctttggggca gcggccaata gcagctttgc tccttcgctt tctgggctca gaggctggga
     1321 aggggtgggt ccgggggcgg gctcaggggc gggctcaggg gcggggcggg cgcccgaagg
     1381 tcctccggag gcccggcatt ctgcacgctt caaaagcgca cgtctgccgc gctgttctcc
     1441 tcttcctcat ctccgggcct ttcgacctgc agcctgttga caattaatca tcggcatagt
     1501 atatcggcat agtataatac gacaaggtga ggaactaaac catgggatcg gccattgaac
     1561 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     1621 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     1681 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg
     1741 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     1801 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     1861 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     1921 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     1981 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     2041 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgatgatc
     2101 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     2161 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     2221 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     2281 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     2341 tctgagggga tcaattctct agagctcgct gatcagcctc gactgtgcct tctagttgcc
     2401 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     2461 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     2521 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     2581 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcga
     2641 ctagagcttg cggaaccctt aatataactt cgtataatgt atgctatacg aagttattag
     2701 gtccctcgag gggatccact agttctagag cggccgccaa ctttattata caaagttggc
     2761 attataaaaa agcattgctt atcaatttgt tgcaacgaac aggtcactat cagtcaaaat
     2821 aaaatcatta tttggagctc catggtagcg ttaacgcggc cgcgatatcc cctatagtga
     2881 gtcgtattac atggtcatag ctgtttcctg gcagctctgg cccgtgtctc aaaatctctg
     2941 atgttacatt gcacaagata aaaatatatc atcatgaaca ataaaactgt ctgcttacat
     3001 aaacagtaat acaaggggtg ttatgagcca tattcaacgg gaaacgtcga ggccgcgatt
     3061 aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca
     3121 atcaggtgcg acaatctatc gcttgtatgg gaagcccgat gcgccagagt tgtttctgaa
     3181 acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct
     3241 gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg
     3301 gttactcacc actgcgatcc ccggaaaaac agcattccag gtattagaag aatatcctga
     3361 ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc
     3421 tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg
     3481 aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt
     3541 tgaacaagtc tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac
     3601 tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat
     3661 tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg
     3721 cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa
     3781 tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct aatcagaatt
     3841 ggttaattgg ttgtaacact ggcagagcat tacgctgact tgacgggacg gcgcaagctc
     3901 atgaccaaaa tcccttaacg tgagttacgc gtcgttccac tgagcgtcag accccgtaga
     3961 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     4021 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     4081 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     4141 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     4201 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     4261 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     4321 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     4381 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     4441 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     4501 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     4561 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     4621 tcacatgtt
//
