LOCUS       sequence-121638-        6513 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses FLAGHA-tagged HuB in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6513)
  TITLE     Identification of the transcriptome-wide mRNA targets of the 
            neuronal ELAVL proteins (unpublished)
REFERENCE   2  (bases 1 to 6513)
  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"
COMMENT     Sequence Label: pFRT-TODestFLAGHA_HuB
FEATURES             Location/Qualifiers
     source          1..6513
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..42
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(86..105)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        277..656
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        657..860
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     811..831
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    862..880
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    883..901
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     911..935
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1035..1058
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1059..1085
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    1101..1125
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2267..2291)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2344..2385
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2465..2482)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2471..2695
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2828..2847)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2979..3026
                     /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             3034..4056
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3034..4056
                     /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    4186..4307
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4223..4242)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4277..4296
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4356..4372)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4356..4372)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4369..4391)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4380..4396
                     /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(4404..4410,4411..4428,4429..4434))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4449..4470
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4587..4604)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4758..5346)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4838..4857)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5517..6308,6309..6377))
                     /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     6140..6159
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6378..6482)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtaagccta tccctaaccc tctcctcggt ctcgattcta cggacggatc gggagatctc
       61 ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
      121 atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta
      181 caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg
      241 cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt
      301 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
      361 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
      421 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
      481 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
      541 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
      601 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
      661 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
      721 gtctccaccc cattgacgtc aatgggagtt tgttttggaa ccaaaatcaa cgggactttc
      781 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
      841 aggtctatat aagcagagct ctccctatca gtgatagaga tctccctatc agtgatagag
      901 atcgtcgacg agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt
      961 ttgacctcca tagaagacac cgggaccgat ccagcctccg gactctagcg tttaaactta
     1021 agcttggtac catggactac aaggacgacg atgacaagta cccttatgac gtgcccgatt
     1081 acgctttgaa ccagatatca acaagtttgt acaaaaaagc aggctccacc atgggaacca
     1141 attcagtcga ctggatccgg atggaaacac aactgtctaa tgggccaact tgcaataaca
     1201 cagccaatgg tccaaccacc ataaacaaca actgttcgtc accagttgac tctgggaaca
     1261 cagaagacag caagaccaac ttaatagtca actaccttcc tcagaacatg acacaggagg
     1321 aactaaagag tctctttggg agcattggtg aaatagagtc ctgtaagctt gtaagagaca
     1381 aaataacagg gcagagcttg ggatatggct ttgtgaacta cattgacccc aaggatgcag
     1441 agaaagctat caacaccctg aatggattga gacttcaaac caaaacaata aaagtttcct
     1501 atgctcgccc aagttcagct tctatcagag atgcaaattt atatgtcagc ggacttccaa
     1561 aaacaatgac ccagaaggag ttggaacagc ttttttcaca atatggacgc attattactt
     1621 ctcgtattct tgtcgaccag gtcactggca tatcaagggg tgtagggttt attcgatttg
     1681 acaagcgaat tgaggcagaa gaagctatca aaggcctaaa tggccagaaa cctcccggtg
     1741 ccacggagcc aatcactgta aagtttgcta ataacccaag ccaaaaaacc aatcaggcca
     1801 tcctttccca gctgtaccag tctccaaaca gaaggtatcc aggaccgcta gctcagcagg
     1861 cacagcgttt taggttggac aatctgctca atatggctta tggagtaaag aggttttctc
     1921 caatgaccat tgacggaatg accagtttgg ctggaattaa tatccctggg caccctggaa
     1981 cagggtggtg tatatttgtg tacaacctgg ctcctgacgc agatgagagt atcctgtggc
     2041 aaatgtttgg gccttttgga gctgtcacca atgtgaaggt catccgtgac tttaacacca
     2101 ataaatgcaa aggttttgga tttgtgacta tgacaaacta tgatgaggct gccatggcga
     2161 tagctagcct caatggatac cgtctgggag acagagtact gcaggtctcc tttaagacaa
     2221 acaaaacgca caaagcctaa tgagcggccg cactcgagat atctagaccc agctttcttg
     2281 tacaaagtgg ttgatatcca gcacagtggc ggccgctcaa gtctagaggg cccgcggttc
     2341 gaaggtaagc ctatccctaa ccctctcctc ggtctcgatt ctacgcgtac ctgatccgcg
     2401 gccgcataga taactgatcc agtgtgcctc gagtctagag ggcccgttta aacccgctga
     2461 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     2521 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     2581 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     2641 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     2701 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     2761 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     2821 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2881 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2941 cccaaaaaac ttgattaggg tgatggttca cgtacctaga agttcctatt ccgaagttcc
     3001 tattctctag aaagtatagg aacttccttg gccaaaaagc ctgaactcac cgcgacgtct
     3061 gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag
     3121 ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta
     3181 aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc
     3241 gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc
     3301 atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct
     3361 gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg
     3421 agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc
     3481 atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc
     3541 agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa
     3601 gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc
     3661 ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc
     3721 aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag
     3781 cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt
     3841 cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag
     3901 ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc
     3961 cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac
     4021 cgacgcccca gcactcgtcc gagggcaaag gaatagcacg tactacgaga tttcgattcc
     4081 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     4141 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     4201 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     4261 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     4321 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     4381 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     4441 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     4501 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     4561 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     4621 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     4681 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4741 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4801 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4861 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4921 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4981 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     5041 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     5101 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     5161 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
     5221 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     5281 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     5341 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     5401 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     5461 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     5521 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     5581 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     5641 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     5701 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     5761 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     5821 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     5881 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     5941 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     6001 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     6061 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     6121 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     6181 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     6241 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     6301 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     6361 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     6421 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     6481 cgcacatttc cccgaaaagt gccacctgac gtc
//
