LOCUS       sequence-115023-        7176 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses GFP-tagged HuD in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7176)
  TITLE     Identification of the transcriptome-wide mRNA targets of the 
            neuronal ELAVL proteins (unpublished)
REFERENCE   2  (bases 1 to 7176)
  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-TODestGFP_HuD
FEATURES             Location/Qualifiers
     source          1..7176
                     /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             join(1032..1034,1035..1037,1038..1748)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1032..1034,1035..1037,1038..1748)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1338..1357)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1685..1706
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    1755..1779
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2930..2954)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3007..3048
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3128..3145)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3134..3358
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3491..3510)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3642..3689
                     /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             3697..4719
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3697..4719
                     /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    4849..4970
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4886..4905)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4940..4959
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5019..5035)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5019..5035)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5032..5054)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5043..5059
                     /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(5067..5073,5074..5091,5092..5097))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5112..5133
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5250..5267)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5421..6009)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5501..5520)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6180..6971,6972..7040))
                     /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     6803..6822
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7041..7145)
                     /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 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     1081 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
     1141 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     1201 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg
     1261 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     1321 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     1381 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     1441 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
     1501 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
     1561 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     1621 ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg
     1681 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     1741 tgtacaagaa gcttacaagt ttgtacaaaa aagcaggctc caccatggga accaattcag
     1801 tcgacatggt tatgataatt agcaccatgg agcctcaggt gtcaaatggt ccgacatcca
     1861 atacaagcaa tggaccctcc agcaacaaca gaaactgtcc ttctcccatg caaacagggg
     1921 caaccacaga tgacagcaaa accaacctca tcgtcaacta tttaccccag aatatgaccc
     1981 aagaagaatt caggagtctc ttcgggagca ttggtgaaat agaatcctgc aaacttgtga
     2041 gagacaaaat tacaggacag agtttagggt atggatttgt taactatatt gatccaaagg
     2101 atgcagagaa agccatcaac actttaaatg gactcagact ccagaccaaa accataaagg
     2161 tctcatatgc ccgtccgagc tctgcctcaa tcagggatgc taacctctat gttagcggcc
     2221 ttcccaaaac catgacccag aaggaactgg agcaactttt ctcgcaatac ggccgtatca
     2281 tcacctcacg aatcctggtt gatcaagtca caggagtgtc cagaggggtg ggattcatcc
     2341 gctttgataa gaggattgag gcagaagaag ccatcaaagg gctgaatggc cagaagccca
     2401 gcggtgctac ggaaccgatt actgtgaagt ttgccaacaa ccccagccag aagtccagcc
     2461 aggccctgct ctcccagctc taccagtccc ctaaccggcg ctacccaggt ccacttcacc
     2521 accaggctca gaggttcagg ctggacaatt tgcttaatat ggcctatggc gtaaagaggt
     2581 tctccccaat taccattgat ggaatgacaa gccttgtggg aatgaacatc cctggtcaca
     2641 caggaactgg gtggtgcatc tttgtctaca acctgtcccc cgattccgat gagagtgtcc
     2701 tctggcagct ctttggcccc tttggagcag tgaacaacgt aaaggtgatt cgtgacttca
     2761 acaccaacaa gtgcaaggga ttcggctttg tcaccatgac caactatgat gaggcggcca
     2821 tggccatcgc cagcctcaac gggtaccgcc tgggagacag agtgttgcaa gtttccttta
     2881 aaaccaacaa agcccacaag tcctgagcgg ccgcactcga gatatctaga cccagctttc
     2941 ttgtacaaag tggttgatat ccagcacagt ggcggccgct caagtctaga gggcccgcgg
     3001 ttcgaaggta agcctatccc taaccctctc ctcggtctcg attctacgcg taccggatcc
     3061 gcggccgcat agataactga tccagtgtgc ctcgagtcta gagggcccgt ttaaacccgc
     3121 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     3181 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     3241 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     3301 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     3361 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     3421 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     3481 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     3541 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     3601 gaccccaaaa aacttgatta gggtgatggt tcacgtacct agaagttcct attccgaagt
     3661 tcctattctc tagaaagtat aggaacttcc ttggccaaaa agcctgaact caccgcgacg
     3721 tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg
     3781 gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg
     3841 gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg
     3901 gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat
     3961 tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc
     4021 gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag
     4081 acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat
     4141 ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc
     4201 gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc
     4261 gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc
     4321 cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc
     4381 gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc
     4441 gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt
     4501 ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg
     4561 cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc
     4621 gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga
     4681 aaccgacgcc ccagcactcg tccgagggca aaggaatagc acgtactacg agatttcgat
     4741 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     4801 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
     4861 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     4921 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
     4981 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     5041 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     5101 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     5161 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     5221 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     5281 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     5341 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     5401 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     5461 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5521 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5581 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5641 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5701 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5761 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5821 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     5881 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5941 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     6001 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     6061 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     6121 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     6181 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     6241 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     6301 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     6361 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     6421 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     6481 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     6541 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     6601 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     6661 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     6721 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     6781 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     6841 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     6901 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     6961 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     7021 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     7081 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     7141 ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
