LOCUS       sequence-213699-        6978 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Doxycycline inducible mammalian retroviral expression of HuR (S221D)
            with FLAG and mKO2 tags.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6978)
  TITLE     Mammalian retroviral expression plasmids based on pBABE family
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6978)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pBABE.puro/TO/Flag/HuR.S221D
FEATURES             Location/Qualifiers
     source          1..6978
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             8..477
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    542..741
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             942..1358
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1288..1310
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1326..1342
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     protein_bind    1368..1386
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1396..1420
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1510..1519
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1519..1542
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1549..2202
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     CDS             1549..2202
                     /codon_start=1
                     /product="humanized monomeric Kusabira-Orange fluorescent 
                     protein"
                     /label=hmKO
                     /note="mammalian codon-optimized"
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     primer_bind     complement(3231..3251)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3236..3565
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3416..3551
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3575..4174
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3575..3594)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4071..4091
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5071..5088)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5242..5830)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5322..5341)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5990..6781,6782..6850))
                     /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     6613..6632
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6851..6955)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgcagcctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca
       61 gggccaagaa cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt
      121 tcctgccccg gctcagggcc aagaacagat ggtccccaga tgcggtccag ccctcagcag
      181 tttctagaga accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc
      241 ttatttgaac taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga
      301 gctcaataaa agagcccaca acccctcact cggggcgcca gtcctccgat tgactgagtc
      361 gcccgggtac ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc
      421 tgttccttgg gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcattt
      481 gggggctcgt ccgggatcgg gagacccctg cccagggacc accgacccac caccgggagg
      541 caagctggcc agcaacttat ctgtgtctgt ccgattgtct agtgtctatg actgatttta
      601 tgcgcctgcg tcggtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa
      661 ctgacgagtt ctgaacaccc ggccgcaacc ctgggagacg tcccagggac tttgggggcc
      721 gtttttgtgg cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg
      781 tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt
      841 cggtttggaa ccgaagccgc gcgtcttgtc tgctgcagcg ctgcagcatc gttctgtgtt
      901 gtctctgtct gactgtgttt ctgtatttgt ctgaaaatta gggccagact gttaccactc
      961 ccttaagttt gaccttaggt cactggaaag atgtcgagcg gatcgctcac aaccagtcgg
     1021 tagatgtcaa gaagagacgt tgggttacct tctgctctgc agaatggcca acctttaacg
     1081 tcggatggcc gcgagacggc acctttaacc gagacctcat cacccaggtt aagatcaagg
     1141 tcttttcacc tggcccgcat ggacacccag accaggtccc ctacatcgtg acctgggaag
     1201 ccttggcttt tgacccccct ccctgggtca agccctttgt acaccctaag cctccgcctc
     1261 ctcttcctcc atccgccccg tctctccccc ttgaacctcc tcgttcgacc ccgcctcgat
     1321 cctcccttta tccagccctc actccttctc taggcgccgg ccggatctcc ctatcagtga
     1381 tagagatcgt cgacgagctc gtttagtgaa ccgtcagatc gcctggagac gccatccacg
     1441 ctgttttgac ctccatagaa gacaccggga ccgatccagc ctccggactc tagcgtttaa
     1501 acttaagctg ccaccatgga ctacaaggac gacgatgaca agaagcttat ggtgagcgtg
     1561 atcaagcccg agatgaagat gaggtactac atggacggct ccgtcaatgg gcatgagttc
     1621 acaatcgagg gtgagggcac aggcagacct tacgagggac atcaggagat gacactgcgc
     1681 gtcacaatgg ccgagggcgg gccaatgcct ttcgccttcg acctggtgtc ccacgtgttc
     1741 tgttacggcc acagagtttt taccaagtac ccagaagaga tcccagacta tttcaagcag
     1801 gcctttcctg agggcctgtc ctgggagagg tccctggagt tcgaggacgg cggctccgcc
     1861 tccgtgagcg cccacatcag cctgaggggc aacaccttct accacaagtc caagttcacc
     1921 ggcgtgaact tccccgccga cggccccatc atgcagaacc agagcgtgga ctgggagccc
     1981 tccaccgaga agatcaccgc cagcgacggc gtgctgaagg gcgacgtgac catgtacctg
     2041 aagctggagg gcggcggcaa ccacaagtgc cagatgaaga ccacctacaa ggccgccaag
     2101 gagatcctgg agatgcccgg cgaccactac atcggccaca ggctggtgag gaagaccgag
     2161 ggcaacatca ccgagcaggt ggaggacgcc gtggcccact ccggtggcgg aggctcgggc
     2221 ggaggtgggt cgggtaccat gtctaatggt tatgaagacc acatggccga agactgcagg
     2281 ggtgacatcg ggagaacgaa tttgatcgtc aactacctcc ctcagaacat gacccaggat
     2341 gagttacgaa gcctgttcag cagcattggt gaagttgaat ctgcaaaact tattcgggat
     2401 aaagtagcag gacacagctt gggctatggc tttgtgaact acgtgaccgc gaaggatgca
     2461 gagagagcga tcaacacgct gaacggcttg aggctccagt caaaaaccat taaggtgtcg
     2521 tatgctcgcc cgagctcaga ggtgatcaaa gacgccaact tgtacatcag cgggctcccg
     2581 cggaccatga cccagaagga cgtagaagac atgttctctc ggtttgggcg gatcatcaac
     2641 tcgcgggtcc tcgtggatca gactacaggt ttgtccagag gggttgcgtt tatccggttt
     2701 gacaaacggt cggaggcaga agaggcaatt accagtttca atggtcataa acccccaggt
     2761 tcctctgagc ccatcacagt gaagtttgca gccaacccca accagaacaa aaacgtggca
     2821 ctcctctcgc agctgtacca ctcgccagcg cgacggttcg gaggccccgt tcaccaccag
     2881 gcgcagagat tcaggttcga ccccatgggc gtcgatcaca tgagcgggct ctctggcgtc
     2941 aacgtgtcag gaaacgcctc ctccggctgg tgcattttca tctacaacct ggggcaggat
     3001 gccgacgagg ggatcctctg gcagatgttt gggccgtttg gtgccgtcac caatgtgaaa
     3061 gtgatccgcg acttcaacac caacaagtgc aaagggtttg gctttgtgac catgacaaac
     3121 tatgaagaag ccgcgatggc catagccagc ctgaacggct accgcctggg ggacaaaatc
     3181 ttacaggttt ccttcaaaac caacaagtcc cacaaataac tcgaccctgt ggaatgtgtg
     3241 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     3301 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     3361 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     3421 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     3481 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     3541 ttttggaggc ctaggctttt gcaaaaagct taccatgacc gagtacaagc ccacggtgcg
     3601 cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga
     3661 ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct
     3721 gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga
     3781 cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc
     3841 cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat
     3901 ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg
     3961 cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga
     4021 ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc
     4081 cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg
     4141 cacctggtgc atgacccgca agcccggtgc ctgacgcccg ccccacgacc cgcagcgccc
     4201 gaccgaaagg agcgcacgac cccatgcatc gataaaataa aagattttat ttagtctcca
     4261 gaaaaagggg ggaatgaaag accccacctg taggtttggc aagctagctt aagtaacgcc
     4321 attttgcaag gcatggaaaa atacataact gagaatagag aagttcagat caaggtcagg
     4381 aacagatgga acagctgaat atgggccaaa caggatatct gtggtaagca gttcctgccc
     4441 cggctcaggg ccaagaacag atggaacagc tgaatatggg ccaaacagga tatctgtggt
     4501 aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc ggtccagccc
     4561 tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc tgaaatgacc
     4621 ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc gcgcttctgc
     4681 tccccgagct caataaaaga gcccacaacc cctcactcgg ggcgccagtc ctccgattga
     4741 ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat ccgacttgtg
     4801 gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca gcggggggca
     4861 gttgcatccg acttgtggtc tcgctgttcc ttgggagggt ctcctctgag tgattgacta
     4921 cccgtcagcg ggggtctttc acatgcagca tgtatcaaaa ttaatttggt tttttttctt
     4981 aagtatttac attaaatggc catagttgca ttaatgaatc ggccaacgcg cggggagagg
     5041 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     5101 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     5161 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     5221 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     5281 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     5341 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     5401 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     5461 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5521 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5581 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5641 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     5701 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5761 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5821 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5881 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5941 gcggccgcaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6001 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6061 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6121 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6181 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     6241 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6301 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     6361 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     6421 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     6481 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     6541 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6601 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6661 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     6721 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     6781 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     6841 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     6901 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     6961 ttccccgaaa agtgccac
//
