LOCUS       sequence-175241-        6475 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express GRB2 with a GFP tag at C terminus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6475)
  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 6475)
  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-GRB2-GFP
FEATURES             Location/Qualifiers
     source          1..6475
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        3..382
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        383..586
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     537..557
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    588..606
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    609..627
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     637..661
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(1431..1433,1434..1436,1437..2150)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1431..1433,1434..1436,1437..2147)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1476..1497)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1737..1756)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2084..2105
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2195..2212)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2201..2425
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2558..2577)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2709..2756
                     /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             2764..3786
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2764..3786
                     /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    3916..4037
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3953..3972)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4007..4026
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4086..4102)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4086..4102)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4099..4121)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4110..4126
                     /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(4134..4140,4141..4158,4159..4164))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4179..4200
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4317..4334)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4488..5076)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4568..4587)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5247..6038,6039..6107))
                     /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     5870..5889
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6108..6212)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6287..6306)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
       61 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      121 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
      181 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
      241 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      301 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      361 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
      421 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
      481 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
      541 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctcc ctatcagtga
      601 tagagatctc cctatcagtg atagagatcg tcgacgagct cgtttagtga accgtcagat
      661 cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg accgatccag
      721 cctccggact ctagcgttta aacttaagct tggtaccgag ctcggatccc accatggaag
      781 ccatcgccaa atatgacttc aaagctactg cagacgacga gctgagcttc aaaagggggg
      841 acatcctcaa ggttttgaac gaagaatgtg atcagaactg gtacaaggca gagcttaatg
      901 gaaaagacgg cttcattccc aagaactaca tagaaatgaa accacatccg tggttttttg
      961 gcaaaatccc cagagccaag gcagaagaaa tgcttagcaa acagcggcac gatggggcct
     1021 ttcttatccg agagagtgag agcgctcctg gggacttctc cctctctgtc aagtttggaa
     1081 acgatgtgca gcacttcaag gtgctccgag atggagccgg gaagtacttc ctctgggtgg
     1141 tgaagttcaa ttctttgaat gagctggtgg attatcacag atctacatct gtctccagaa
     1201 accagcagat attcctgcgg gacatagaac aggtgccaca gcagccgaca tacgtccagg
     1261 ccctctttga ctttgatccc caggaggatg gagagctggg cttccgccgg ggagatttta
     1321 tccatgtcat ggataactca gaccccaact ggtggaaagg agcttgccac gggcagaccg
     1381 gcatgtttcc ccgcaattat gtcacccccg tgaaccggaa cgtcgatatc atggtgagca
     1441 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     1501 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     1561 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     1621 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
     1681 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     1741 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     1801 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     1861 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     1921 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     1981 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     2041 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     2101 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtag gcggccgctc
     2161 gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     2221 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     2281 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     2341 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     2401 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     2461 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     2521 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     2581 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     2641 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     2701 cgtacctaga agttcctatt ccgaagttcc tattctctag aaagtatagg aacttccttg
     2761 gccaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac
     2821 agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat
     2881 gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat
     2941 cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt
     3001 ggggaattca gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg
     3061 caagacctgc ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat
     3121 gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga
     3181 atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat
     3241 cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag
     3301 ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc
     3361 tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg
     3421 atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct
     3481 tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg
     3541 cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac
     3601 ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga
     3661 gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc
     3721 tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag
     3781 gaatagcacg tactacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     3841 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     3901 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     3961 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     4021 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
     4081 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     4141 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     4201 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4261 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     4321 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     4381 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     4441 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     4501 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     4561 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     4621 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4681 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4741 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4801 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4861 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4921 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4981 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     5041 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     5101 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     5161 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     5221 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     5281 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     5341 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     5401 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     5461 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     5521 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     5581 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     5641 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     5701 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     5761 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     5821 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     5881 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     5941 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     6001 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     6061 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     6121 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     6181 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     6241 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
     6301 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
     6361 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
     6421 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcg
//
