LOCUS       sequence-111834-        7172 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses GFP-tagged HuC in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7172)
  TITLE     Identification of the transcriptome-wide mRNA targets of the 
            neuronal ELAVL proteins (unpublished)
REFERENCE   2  (bases 1 to 7172)
  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_HuC
FEATURES             Location/Qualifiers
     source          1..7172
                     /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(1077..1098)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     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(2926..2950)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3003..3044
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3124..3141)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3130..3354
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3487..3506)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3638..3685
                     /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             3693..4715
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3693..4715
                     /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    4845..4966
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4882..4901)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4936..4955
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5015..5031)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5015..5031)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5028..5050)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5039..5055
                     /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(5063..5069,5070..5087,5088..5093))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5108..5129
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5246..5263)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5417..6005)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5497..5516)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6176..6967,6968..7036))
                     /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     6799..6818
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7037..7141)
                     /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 cactcagata ctgggggcca tggagtctca ggtggggggg ggcccggccg
     1861 gcccggccct gcccaacggg ccactccttg gtacaaatgg agccactgac gacagcaaga
     1921 ccaacctcat cgtcaactac ctgccccaga acatgaccca ggatgagttc aagagtctct
     1981 tcggcagcat tggcgacatc gagtcctgca agttggttcg ggacaagatc acagggcaga
     2041 gccttggcta cgggtttgtg aactattctg accccaatga tgcagacaaa gccatcaaca
     2101 ccctcaacgg cctcaaatta cagacgaaga ccatcaaggt gtcctatgcc agacccagtt
     2161 cagcatccat ccgggatgct aacctgtacg tcagcgggct ccccaagacc atgagccaga
     2221 aagagatgga gcagctcttc tcccagtacg gccgcatcat cacgtcccgc atcctggtgg
     2281 accaggtcac aggtgtctct cggggtgtgg gattcatccg ctttgacaag aggattgagg
     2341 ccgaagaggc tatcaaagga ctgaatgggc agaagccgct gggcgcagct gagcccatca
     2401 cagtcaagtt cgcgaacaac ccaagtcaga agacggggca ggcgctgctc acccacctct
     2461 accagtcatc cgcccggcgc tacgcaggcc ccctacacca tcagacccag cgtttccggc
     2521 tggacaattt gctcaacatg gcctacggcg tcaagagtcc cctgtcgctc atcgccaggt
     2581 tctcgccgat cgccatcgat ggtatgagcg gcctggcggg cgtgggcctg tcggggggcg
     2641 cggcgggcgc cggctggtgc atcttcgtgt acaacctgtc accggaggca gacgagagcg
     2701 tgctgtggca gctgttcggg ccttttgggg cagtcaccaa cgtcaaggtc atccgtgatt
     2761 tcaccaccaa caagtgcaag ggtttcggct tcgtgaccat gaccaactat gacgaggcgg
     2821 ccatggccat cgccagcctg aacggctatc gcctgggcga gcgcgtgctg caggtctcct
     2881 tcaagaccag caaacagcac aaggcgtgaa agggtgggcg cgccgaccca gctttcttgt
     2941 acaaagtggt tgatatccag cacagtggcg gccgctcaag tctagagggc ccgcggttcg
     3001 aaggtaagcc tatccctaac cctctcctcg gtctcgattc tacgcgtacc ggatccgcgg
     3061 ccgcatagat aactgatcca gtgtgcctcg agtctagagg gcccgtttaa acccgctgat
     3121 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     3181 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     3241 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     3301 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     3361 aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat
     3421 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     3481 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     3541 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     3601 ccaaaaaact tgattagggt gatggttcac gtacctagaa gttcctattc cgaagttcct
     3661 attctctaga aagtatagga acttccttgg ccaaaaagcc tgaactcacc gcgacgtctg
     3721 tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag ctctcggagg
     3781 gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa
     3841 atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatcggccg
     3901 cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg acctattgca
     3961 tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg
     4021 ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc ggccgatctt agccagacga
     4081 gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca
     4141 tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca
     4201 gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag
     4261 tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca
     4321 taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca
     4381 acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc
     4441 ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc cgcattggtc
     4501 ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg
     4561 gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc
     4621 gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc
     4681 gacgccccag cactcgtccg agggcaaagg aatagcacgt actacgagat ttcgattcca
     4741 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     4801 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     4861 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4921 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     4981 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     5041 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     5101 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     5161 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5221 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     5281 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5341 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5401 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     5461 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5521 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5581 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     5641 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5701 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5761 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5821 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     5881 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5941 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     6001 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     6061 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     6121 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     6181 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     6241 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     6301 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     6361 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6421 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6481 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6541 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6601 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6661 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6721 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6781 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6841 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6901 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6961 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     7021 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     7081 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     7141 gcacatttcc ccgaaaagtg ccacctgacg tc
//
