LOCUS       sequence-207756-        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  JOURNAL   Unpublished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            "Unpublished"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"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      983..992
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             992..1015
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1022..1717
                     /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(2748..2765)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2754..2978
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3111..3130)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3262..3309
                     /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             3317..4339
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3317..4339
                     /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    4469..4590
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4506..4525)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4560..4579
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4639..4655)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4639..4655)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4652..4674)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4663..4679
                     /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(4687..4693,4694..4711,4712..4717))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4732..4753
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4870..4887)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5041..5629)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5121..5140)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5800..6591,6592..6660))
                     /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     6423..6442
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6661..6765)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag ctgccaccat ggactacaag gacgacgatg acaagaagct
     1021 tatggagagc gacgagagcg gcctgcccgc catggagatc gagtgccgca tcaccggcac
     1081 cctgaacggc gtggagttcg agctggtggg cggcggagag ggcacccccg agcagggccg
     1141 catgaccaac aagatgaaga gcaccaaagg cgccctgacc ttcagcccct acctgctgag
     1201 ccacgtgatg ggctacggct tctaccactt cggcacctac cccagcggct acgagaaccc
     1261 cttcctgcac gccatcaaca acggcggcta caccaacacc cgcatcgaga agtacgagga
     1321 cggcggcgtg ctgcacgtga gcttcagcta ccgctacgag gccggccgcg tgatcggcga
     1381 cttcaaggtg atgggcaccg gcttccccga ggacagcgtg atcttcaccg acaagatcat
     1441 ccgcagcaac gccaccgtgg agcacctgca ccccatgggc gataacgatc tggatggcag
     1501 cttcacccgc accttcagcc tgcgcgacgg cggctactac agctccgtgg tggacagcca
     1561 catgcacttc aagagcgcca tccaccccag catcctgcag aacgggggcc ccatgttcgc
     1621 cttccgccgc gtggaggagg atcacagcaa caccgagctg ggcatcgtgg agtaccagca
     1681 cgccttcaag accccggatg cagatgccgg tgaagaaaga gttggtacca tgtctaatgg
     1741 ttatgaagac cacatggccg aagactgcag gggtgacatc gggagaacga atttgatcgt
     1801 caactacctc cctcagaaca tgacccagga tgagttacga agcctgttca gcagcattgg
     1861 tgaagttgaa tctgcaaaac ttattcggga taaagtagca ggacacagct tgggctatgg
     1921 ctttgtgaac tacgtgaccg cgaaggatgc agagagagcg atcaacacgc tgaacggctt
     1981 gaggctccag tcaaaaacca ttaaggtgtc gtatgctcgc ccgagctcag aggtgatcaa
     2041 agacgccaac ttgtacatca gcgggctccc gcggaccatg acccagaagg acgtagaaga
     2101 catgttctct cggtttgggc ggatcatcaa ctcgcgggtc ctcgtggatc agactacagg
     2161 tttgtccaga ggggttgcgt ttatccggtt tgacaaacgg tcggaggcag aagaggcaat
     2221 taccagtttc aatggtcata aacccccagg ttcctctgag cccatcacag tgaagtttgc
     2281 agccaacccc aaccagaaca aaaacgtggc actcctctcg cagctgtacc actcgccagc
     2341 gcgacggttc ggaggccccg ttcaccacca ggcgcagaga ttcaggttct cccccatggg
     2401 cgtcgatcac atgagcgggc tctctggcgt caacgtgcca ggaaacgcct cctccggctg
     2461 gtgcattttc atctacaacc tggggcagga tgccgacgag gggatcctct ggcagatgtt
     2521 tgggccgttt ggtgccgtca ccaatgtgaa agtgatccgc gacttcaaca ccaacaagtg
     2581 caaagggttt ggctttgtga ccatgacaaa ctatgaagaa gccgcgatgg ccatagccag
     2641 cctgaacggc taccgcctgg gggacaaaat cttacaggtt tccttcaaaa ccaacaagtc
     2701 ccacaaataa ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc
     2761 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     2821 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     2881 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     2941 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     3001 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     3061 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     3121 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     3181 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     3241 gggtgatggt tcacgtacct agaagttcct attccgaagt tcctattctc tagaaagtat
     3301 aggaacttcc ttggccaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat
     3361 cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc
     3421 tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg
     3481 tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga
     3541 agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca
     3601 gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc
     3661 ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt
     3721 cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga
     3781 tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca
     3841 ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca
     3901 cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga
     3961 ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag
     4021 gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct
     4081 tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca
     4141 gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat
     4201 cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt
     4261 ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg
     4321 tccgagggca aaggaatagc acgtactacg agatttcgat tccaccgccg ccttctatga
     4381 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     4441 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     4501 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     4561 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     4621 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     4681 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     4741 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     4801 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     4861 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     4921 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     4981 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     5041 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     5101 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     5161 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     5221 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     5281 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     5341 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     5401 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     5461 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     5521 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     5581 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     5641 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     5701 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     5761 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     5821 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     5881 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     5941 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     6001 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     6061 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     6121 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     6181 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     6241 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     6301 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     6361 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     6421 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     6481 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     6541 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     6601 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     6661 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     6721 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     6781 agtgccacct gacgtc
//
