LOCUS       sequence-213402-        6796 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of turboGFP-HuR (wild type) with FLAG tag, 
            Flp-In T-Rex system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6796)
  TITLE     Mammalian expression plasmids based on pcDNA5/FRT/TO family
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6796)
  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            "Unpublished"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-tGFP-HuR.wt
FEATURES             Location/Qualifiers
     source          1..6796
                     /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             788..1483
                     /codon_start=1
                     /product="improved green fluorescent protein from 
                     Pontellina plumata (Evdokimov et al., 2006)"
                     /label=TurboGFP
                     /note="mammalian codon-optimized"
                     /translation="MESDESGLPAMEIECRITGTLNGVEFELVGGGEGTPEQGRMTNKM
                     KSTKGALTFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVL
                     HVSFSYRYEAGRVIGDFKVMGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNDLDGSFTR
                     TFSLRDGGYYSSVVDSHMHFKSAIHPSILQNGGPMFAFRRVEEDHSNTELGIVEYQHAF
                     KTPDADAGEE"
     primer_bind     complement(2514..2531)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2520..2744
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2877..2896)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3028..3075
                     /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             3083..4105
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3083..4105
                     /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    4235..4356
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4272..4291)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4326..4345
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4405..4421)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4405..4421)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4418..4440)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4429..4445
                     /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(4453..4459,4460..4477,4478..4483))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4498..4519
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4636..4653)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4807..5395)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4887..4906)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5566..6357,6358..6426))
                     /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     6189..6208
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6427..6531)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6606..6625)
                     /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 gaagcttatg gagagcgacg agagcggcct gcccgccatg gagatcgagt gccgcatcac
      841 cggcaccctg aacggcgtgg agttcgagct ggtgggcggc ggagagggca cccccgagca
      901 gggccgcatg accaacaaga tgaagagcac caaaggcgcc ctgaccttca gcccctacct
      961 gctgagccac gtgatgggct acggcttcta ccacttcggc acctacccca gcggctacga
     1021 gaaccccttc ctgcacgcca tcaacaacgg cggctacacc aacacccgca tcgagaagta
     1081 cgaggacggc ggcgtgctgc acgtgagctt cagctaccgc tacgaggccg gccgcgtgat
     1141 cggcgacttc aaggtgatgg gcaccggctt ccccgaggac agcgtgatct tcaccgacaa
     1201 gatcatccgc agcaacgcca ccgtggagca cctgcacccc atgggcgata acgatctgga
     1261 tggcagcttc acccgcacct tcagcctgcg cgacggcggc tactacagct ccgtggtgga
     1321 cagccacatg cacttcaaga gcgccatcca ccccagcatc ctgcagaacg ggggccccat
     1381 gttcgccttc cgccgcgtgg aggaggatca cagcaacacc gagctgggca tcgtggagta
     1441 ccagcacgcc ttcaagaccc cggatgcaga tgccggtgaa gaaagagttg gtaccatgtc
     1501 taatggttat gaagaccaca tggccgaaga ctgcaggggt gacatcggga gaacgaattt
     1561 gatcgtcaac tacctccctc agaacatgac ccaggatgag ttacgaagcc tgttcagcag
     1621 cattggtgaa gttgaatctg caaaacttat tcgggataaa gtagcaggac acagcttggg
     1681 ctatggcttt gtgaactacg tgaccgcgaa ggatgcagag agagcgatca acacgctgaa
     1741 cggcttgagg ctccagtcaa aaaccattaa ggtgtcgtat gctcgcccga gctcagaggt
     1801 gatcaaagac gccaacttgt acatcagcgg gctcccgcgg accatgaccc agaaggacgt
     1861 agaagacatg ttctctcggt ttgggcggat catcaactcg cgggtcctcg tggatcagac
     1921 tacaggtttg tccagagggg ttgcgtttat ccggtttgac aaacggtcgg aggcagaaga
     1981 ggcaattacc agtttcaatg gtcataaacc cccaggttcc tctgagccca tcacagtgaa
     2041 gtttgcagcc aaccccaacc agaacaaaaa cgtggcactc ctctcgcagc tgtaccactc
     2101 gccagcgcga cggttcggag gccccgttca ccaccaggcg cagagattca ggttctcccc
     2161 catgggcgtc gatcacatga gcgggctctc tggcgtcaac gtgccaggaa acgcctcctc
     2221 cggctggtgc attttcatct acaacctggg gcaggatgcc gacgagggga tcctctggca
     2281 gatgtttggg ccgtttggtg ccgtcaccaa tgtgaaagtg atccgcgact tcaacaccaa
     2341 caagtgcaaa gggtttggct ttgtgaccat gacaaactat gaagaagccg cgatggccat
     2401 agccagcctg aacggctacc gcctggggga caaaatctta caggtttcct tcaaaaccaa
     2461 caagtcccac aaataactcg agtctagagg gcccgtttaa acccgctgat cagcctcgac
     2521 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     2581 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     2641 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     2701 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     2761 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     2821 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     2881 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     2941 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3001 tgattagggt gatggttcac gtacctagaa gttcctattc cgaagttcct attctctaga
     3061 aagtatagga acttccttgg ccaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt
     3121 tctgatcgaa aagttcgaca gcgtctccga cctgatgcag ctctcggagg gcgaagaatc
     3181 tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa atagctgcgc
     3241 cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatcggccg cgctcccgat
     3301 tccggaagtg cttgacattg gggaattcag cgagagcctg acctattgca tctcccgccg
     3361 tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc
     3421 ggtcgcggag gccatggatg cgatcgctgc ggccgatctt agccagacga gcgggttcgg
     3481 cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca tatgcgcgat
     3541 tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca gtgcgtccgt
     3601 cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag tccggcacct
     3661 cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca taacagcggt
     3721 cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcttctt
     3781 ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc ggaggcatcc
     3841 ggagcttgca ggatcgccgc ggctccgggc gtatatgctc cgcattggtc ttgaccaact
     3901 ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga
     3961 cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc
     4021 ggccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc gacgccccag
     4081 cactcgtccg agggcaaagg aatagcacgt actacgagat ttcgattcca ccgccgcctt
     4141 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     4201 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
     4261 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     4321 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
     4381 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     4441 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     4501 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     4561 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     4621 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     4681 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     4741 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     4801 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     4861 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     4921 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     4981 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     5041 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     5101 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     5161 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     5221 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     5281 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     5341 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     5401 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     5461 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     5521 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     5581 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     5641 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     5701 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     5761 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     5821 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     5881 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     5941 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     6001 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     6061 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     6121 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     6181 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     6241 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     6301 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     6361 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     6421 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     6481 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     6541 ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact
     6601 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg
     6661 ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc
     6721 gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg
     6781 gccagatata cgcgtt
//
