LOCUS       sequence-254685-        5960 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Doxycycline inducible mammalian retroviral expression of mKO2 with 
            FLAG tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5960)
  TITLE     Mammalian retroviral expression plasmids based on pBABE family
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5960)
  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            "Unpublished"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/mKO2
FEATURES             Location/Qualifiers
     source          1..5960
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..470
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    535..734
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             935..1351
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1281..1303
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1319..1335
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     protein_bind    1361..1379
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1389..1413
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1503..1512
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1512..1535
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1545..2198
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     CDS             1545..2198
                     /codon_start=1
                     /product="humanized monomeric Kusabira-Orange fluorescent 
                     protein"
                     /label=hmKO
                     /note="mammalian codon-optimized"
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     primer_bind     complement(2283..2303)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2288..2617
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2468..2603
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2627..3226
                     /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(2627..2646)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3123..3143
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3447..3916
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(4046..4063)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4217..4805)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4297..4316)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4965..5756,5757..5825))
                     /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     5588..5607
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5826..5930)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa
       61 gaacagatgg aacagctgaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc
      121 ccggctcagg gccaagaaca gatggtcccc agatgcggtc cagccctcag cagtttctag
      181 agaaccatca gatgtttcca gggtgcccca aggacctgaa atgaccctgt gccttatttg
      241 aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat
      301 aaaagagccc acaacccctc actcggggcg ccagtcctcc gattgactga gtcgcccggg
      361 tacccgtgta tccaataaac cctcttgcag ttgcatccga cttgtggtct cgctgttcct
      421 tgggagggtc tcctctgagt gattgactac ccgtcagcgg gggtctttca tttgggggct
      481 cgtccgggat cgggagaccc ctgcccaggg accaccgacc caccaccggg aggcaagctg
      541 gccagcaact tatctgtgtc tgtccgattg tctagtgtct atgactgatt ttatgcgcct
      601 gcgtcggtac tagttagcta actagctctg tatctggcgg acccgtggtg gaactgacga
      661 gttctgaaca cccggccgca accctgggag acgtcccagg gactttgggg gccgtttttg
      721 tggcccgacc tgaggaaggg agtcgatgtg gaatccgacc ccgtcaggat atgtggttct
      781 ggtaggagac gagaacctaa aacagttccc gcctccgtct gaatttttgc tttcggtttg
      841 gaaccgaagc cgcgcgtctt gtctgctgca gcgctgcagc atcgttctgt gttgtctctg
      901 tctgactgtg tttctgtatt tgtctgaaaa ttagggccag actgttacca ctcccttaag
      961 tttgacctta ggtcactgga aagatgtcga gcggatcgct cacaaccagt cggtagatgt
     1021 caagaagaga cgttgggtta ccttctgctc tgcagaatgg ccaaccttta acgtcggatg
     1081 gccgcgagac ggcaccttta accgagacct catcacccag gttaagatca aggtcttttc
     1141 acctggcccg catggacacc cagaccaggt cccctacatc gtgacctggg aagccttggc
     1201 ttttgacccc cctccctggg tcaagccctt tgtacaccct aagcctccgc ctcctcttcc
     1261 tccatccgcc ccgtctctcc cccttgaacc tcctcgttcg accccgcctc gatcctccct
     1321 ttatccagcc ctcactcctt ctctaggcgc cggccggatc tccctatcag tgatagagat
     1381 cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt
     1441 gacctccata gaagacaccg ggaccgatcc agcctccgga ctctagcgtt taaacttaag
     1501 ctgccaccat ggactacaag gacgacgatg acaagaagaa gcttatggtg agcgtgatca
     1561 agcccgagat gaagatgagg tactacatgg acggctccgt caatgggcat gagttcacaa
     1621 tcgagggtga gggcacaggc agaccttacg agggacatca ggagatgaca ctgcgcgtca
     1681 caatggccga gggcgggcca atgcctttcg ccttcgacct ggtgtcccac gtgttctgtt
     1741 acggccacag agtttttacc aagtacccag aagagatccc agactatttc aagcaggcct
     1801 ttcctgaggg cctgtcctgg gagaggtccc tggagttcga ggacggcggc tccgcctccg
     1861 tgagcgccca catcagcctg aggggcaaca ccttctacca caagtccaag ttcaccggcg
     1921 tgaacttccc cgccgacggc cccatcatgc agaaccagag cgtggactgg gagccctcca
     1981 ccgagaagat caccgccagc gacggcgtgc tgaagggcga cgtgaccatg tacctgaagc
     2041 tggagggcgg cggcaaccac aagtgccaga tgaagaccac ctacaaggcc gccaaggaga
     2101 tcctggagat gcccggcgac cactacatcg gccacaggct ggtgaggaag accgagggca
     2161 acatcaccga gcaggtggag gacgccgtgg cccactccgg tggcggaggc tcgggcggag
     2221 gtgggtcggg taccgagctc ggatccgaat tcctgcagtc tagagcggcc gctcgaccct
     2281 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     2341 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     2401 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     2461 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     2521 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     2581 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag cttaccatga ccgagtacaa
     2641 gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc
     2701 cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg
     2761 ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg
     2821 ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg
     2881 ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc
     2941 gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct
     3001 ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct
     3061 ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc
     3121 ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc
     3181 cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgacgcc cgccccacga
     3241 cccgcagcgc ccgaccgaaa ggagcgcacg accccatgca tcgataaaat aaaagatttt
     3301 atttagtctc cagaaaaagg ggggaatgaa agaccccacc tgtaggtttg gcaagctagc
     3361 ttaagtaacg ccattttgca aggcatggaa aaatacataa ctgagaatag agaagttcag
     3421 atcaaggtca ggaacagatg gaacagctga atatgggcca aacaggatat ctgtggtaag
     3481 cagttcctgc cccggctcag ggccaagaac agatggaaca gctgaatatg ggccaaacag
     3541 gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg gtccccagat
     3601 gcggtccagc cctcagcagt ttctagagaa ccatcagatg tttccagggt gccccaagga
     3661 cctgaaatga ccctgtgcct tatttgaact aaccaatcag ttcgcttctc gcttctgttc
     3721 gcgcgcttct gctccccgag ctcaataaaa gagcccacaa cccctcactc ggggcgccag
     3781 tcctccgatt gactgagtcg cccgggtacc cgtgtatcca ataaaccctc ttgcagttgc
     3841 atccgacttg tggtctcgct gttccttggg agggtctcct ctgagtgatt gactacccgt
     3901 cagcgggggt ctttcacatg cagcatgtat caaaattaat ttggtttttt ttcttaagta
     3961 tttacattaa atggccatag ttgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4021 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4081 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4141 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4201 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4261 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4321 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     4381 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     4441 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     4501 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     4561 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     4621 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     4681 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     4741 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     4801 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     4861 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttgcggc
     4921 cgcaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     4981 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     5041 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     5101 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     5161 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     5221 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     5281 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     5341 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     5401 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     5461 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     5521 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     5581 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     5641 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     5701 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     5761 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     5821 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     5881 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     5941 cgaaaagtgc cacctgcagc
//
