LOCUS       sequence-213400-        6688 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of mKO2-HuR (DelHNS) with FLAG tag, Flp-In 
            T-Rex system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6688)
  TITLE     Mammalian expression plasmids based on pcDNA5/FRT/TO family
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6688)
  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            "Unpublished"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: pcDNA5/FRT/TO/Flag-mKO2-HuR.DelHNS
FEATURES             Location/Qualifiers
     source          1..6688
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    586..604
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    607..625
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     635..659
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      749..758
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             758..781
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             791..1444
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     CDS             791..1444
                     /codon_start=1
                     /product="humanized monomeric Kusabira-Orange fluorescent 
                     protein"
                     /label=hmKO
                     /note="mammalian codon-optimized"
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     primer_bind     complement(2406..2423)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2412..2636
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2769..2788)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2920..2967
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             2975..3997
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2975..3997
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4127..4248
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4164..4183)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4218..4237
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4297..4313)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4297..4313)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4310..4332)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4321..4337
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4345..4351,4352..4369,4370..4375))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4390..4411
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4528..4545)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4699..5287)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4779..4798)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5458..6249,6250..6318))
                     /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     6081..6100
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6319..6423)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6498..6517)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctccct atcagtgata
      601 gagatctccc tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg
      661 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
      721 tccggactct agcgtttaaa cttaagctgc caccatggac tacaaggacg acgatgacaa
      781 gaagaagctt atggtgagcg tgatcaagcc cgagatgaag atgaggtact acatggacgg
      841 ctccgtcaat gggcatgagt tcacaatcga gggtgagggc acaggcagac cttacgaggg
      901 acatcaggag atgacactgc gcgtcacaat ggccgagggc gggccaatgc ctttcgcctt
      961 cgacctggtg tcccacgtgt tctgttacgg ccacagagtt tttaccaagt acccagaaga
     1021 gatcccagac tatttcaagc aggcctttcc tgagggcctg tcctgggaga ggtccctgga
     1081 gttcgaggac ggcggctccg cctccgtgag cgcccacatc agcctgaggg gcaacacctt
     1141 ctaccacaag tccaagttca ccggcgtgaa cttccccgcc gacggcccca tcatgcagaa
     1201 ccagagcgtg gactgggagc cctccaccga gaagatcacc gccagcgacg gcgtgctgaa
     1261 gggcgacgtg accatgtacc tgaagctgga gggcggcggc aaccacaagt gccagatgaa
     1321 gaccacctac aaggccgcca aggagatcct ggagatgccc ggcgaccact acatcggcca
     1381 caggctggtg aggaagaccg agggcaacat caccgagcag gtggaggacg ccgtggccca
     1441 ctccggtggc ggaggctcgg gcggaggtgg gtcgggtacc atgtctaatg gttatgaaga
     1501 ccacatggcc gaagactgca ggggtgacat cgggagaacg aatttgatcg tcaactacct
     1561 ccctcagaac atgacccagg atgagttacg aagcctgttc agcagcattg gtgaagttga
     1621 atctgcaaaa cttattcggg ataaagtagc aggacacagc ttgggctatg gctttgtgaa
     1681 ctacgtgacc gcgaaggatg cagagagagc gatcaacacg ctgaacggct tgaggctcca
     1741 gtcaaaaacc attaaggtgt cgtatgctcg cccgagctca gaggtgatca aagacgccaa
     1801 cttgtacatc agcgggctcc cgcggaccat gacccagaag gacgtagaag acatgttctc
     1861 tcggtttggg cggatcatca actcgcgggt cctcgtggat cagactacag gtttgtccag
     1921 aggggttgcg tttatccggt ttgacaaacg gtcggaggca gaagaggcaa ttaccagttt
     1981 caatggtcat aaacccccag gttcctctga gcccatcaca gtgaagtttg cagccaaccc
     2041 caaccagaac aaaaacgtgg cactcctctc gcagctgtac cactcgccag cgaagcttgg
     2101 aaacgcctcc tccggctggt gcattttcat ctacaacctg gggcaggatg ccgacgaggg
     2161 gatcctctgg cagatgtttg ggccgtttgg tgccgtcacc aatgtgaaag tgatccgcga
     2221 cttcaacacc aacaagtgca aagggtttgg ctttgtgacc atgacaaact atgaagaagc
     2281 cgcgatggcc atagccagcc tgaacggcta ccgcctgggg gacaaaatct tacaggtttc
     2341 cttcaaaacc aacaagtccc acaaataact cgagtctaga gggcccgttt aaacccgctg
     2401 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     2461 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     2521 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     2581 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     2641 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     2701 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     2761 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     2821 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     2881 ccccaaaaaa cttgattagg gtgatggttc acgtacctag aagttcctat tccgaagttc
     2941 ctattctcta gaaagtatag gaacttcctt ggccaaaaag cctgaactca ccgcgacgtc
     3001 tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc gacctgatgc agctctcgga
     3061 gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt
     3121 aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc
     3181 cgcgctcccg attccggaag tgcttgacat tggggaattc agcgagagcc tgacctattg
     3241 catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc
     3301 tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc ttagccagac
     3361 gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt
     3421 catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt
     3481 cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga
     3541 agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg
     3601 cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc
     3661 caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga
     3721 gcggaggcat ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg
     3781 tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca
     3841 gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc
     3901 ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa
     3961 ccgacgcccc agcactcgtc cgagggcaaa ggaatagcac gtactacgag atttcgattc
     4021 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     4081 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
     4141 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     4201 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
     4261 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     4321 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     4381 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     4441 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4501 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     4561 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     4621 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     4681 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     4741 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     4801 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     4861 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     4921 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     4981 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     5041 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     5101 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     5161 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5221 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5281 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     5341 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     5401 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     5461 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     5521 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     5581 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     5641 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     5701 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     5761 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     5821 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     5881 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     5941 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     6001 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     6061 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     6121 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     6181 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     6241 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     6301 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     6361 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     6421 gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc
     6481 ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc
     6541 cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg
     6601 caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt
     6661 cgcgatgtac gggccagata tacgcgtt
//
