LOCUS       sequence-175219-        6045 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express FNTA with a FLAG tag at C terminus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6045)
  AUTHORS   Xu Y, Anderson DE, Ye Y
  TITLE     The HECT domain ubiquitin ligase HUWE1 targets unassembled soluble 
            proteins for degradation.
  JOURNAL   Cell Discov. 2016 Nov 8;2:16040. eCollection 2016.
  PUBMED    27867533
REFERENCE   2  (bases 1 to 6045)
  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: "Cell 
            Discov."; date: "2016-11-8"; volume: "2"; pages: "16040. eCollection
            2016"
COMMENT     Sequence Label: pcDNA5/FRT/TO-FNTA-FLAG
FEATURES             Location/Qualifiers
     source          1..6045
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        16..395
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        396..599
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     550..570
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    601..619
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    622..640
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     650..674
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      769..778
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1707..1730
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1778..1795)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1784..2008
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2141..2160)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2292..2339
                     /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             2347..3369
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2347..3369
                     /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    3499..3620
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3536..3555)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3590..3609
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3669..3685)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3669..3685)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3682..3704)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3693..3709
                     /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(3717..3723,3724..3741,3742..3747))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3762..3783
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3900..3917)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4071..4659)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4151..4170)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4830..5621,5622..5690))
                     /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     5453..5472
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5691..5795)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5870..5889)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt
       61 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
      121 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
      181 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
      241 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
      301 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
      361 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
      421 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
      481 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
      541 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc
      601 tccctatcag tgatagagat ctccctatca gtgatagaga tcgtcgacga gctcgtttag
      661 tgaaccgtca gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagacacc
      721 gggaccgatc cagcctccgg actctagcgt ttaaacttaa gcttaccgcc atggcggcca
      781 ccgagggggt cggggaggct gcgcaagggg gcgagcccgg gcagccggcg caacccccgc
      841 cccagccgca cccaccgccg ccccagcagc agcacaagga agagatggcg gccgaggctg
      901 gggaagccgt ggcgtccccc atggacgacg ggtttgtgag cctggactcg ccctcctatg
      961 tcctgtacag gcatttccgg agagttcttt tgaagtcact tcagaaggat ctacatgagg
     1021 aaatgaacta catcactgca ataattgagg agcagcccaa aaactatcaa gtttggcatc
     1081 ataggcgagt attagtggaa tggctaagag atccatctca ggagcttgaa tttattgctg
     1141 atattcttaa tcaggatgca aagaattatc atgcctggca gcatcgacaa tgggttattc
     1201 aggaatttaa actttgggat aatgagctgc agtatgtgga ccaacttctg aaagaggatg
     1261 tgagaaataa ctctgtctgg aaccaaagat acttcgttat ttctaacacc actggctaca
     1321 atgatcgtgc tgtattggag agagaagtcc aatacactct ggaaatgatt aaactagtac
     1381 cacataatga aagtgcatgg aactatttga aagggatttt gcaggatcgt ggtctttcca
     1441 aatatcctaa tctgttaaat caattacttg atttacaacc aagtcatagt tccccctacc
     1501 taattgcctt tcttgtggat atctatgaag acatgctaga aaatcagtgt gacaataagg
     1561 aagacattct taataaagca ttagagttat gtgaaatcct agctaaagaa aaggacacta
     1621 taagaaagga atattggaga tacattggaa gatcccttca aagcaaacac agcacagaaa
     1681 atgactcacc aacaaatgta cagcaagatt acaaggatga tgatgataag taggcggccg
     1741 ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc
     1801 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     1861 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     1921 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     1981 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     2041 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     2101 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     2161 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     2221 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     2281 tcacgtacct agaagttcct attccgaagt tcctattctc tagaaagtat aggaacttcc
     2341 ttggccaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc
     2401 gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc
     2461 gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa
     2521 gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac
     2581 attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg
     2641 ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc ggaggccatg
     2701 gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa
     2761 ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg
     2821 tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat
     2881 gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc
     2941 ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag
     3001 gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg
     3061 gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg
     3121 ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt
     3181 gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc
     3241 ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat
     3301 ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca
     3361 aaggaatagc acgtactacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     3421 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     3481 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     3541 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     3601 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg
     3661 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     3721 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     3781 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     3841 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     3901 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     3961 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     4021 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     4081 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     4141 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     4201 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     4261 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4321 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4381 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4441 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4501 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     4561 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     4621 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     4681 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     4741 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     4801 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     4861 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     4921 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     4981 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     5041 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     5101 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     5161 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     5221 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     5281 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     5341 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     5401 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     5461 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     5521 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     5581 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     5641 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     5701 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     5761 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     5821 gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct
     5881 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
     5941 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga
     6001 atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acggg
//
