LOCUS       sequence-149706-        7157 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses under the CAG promoter RandE3, a synthetic E3 ligase with 
            random residues in its binding domain; control construct for GFE3..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7157)
  AUTHORS   Gross GG, Straub C, Perez-Sanchez J, Dempsey WP, Junge JA, Roberts 
            RW, Trinh LA, Fraser SE, De Koninck Y, De Koninck P, Sabatini BL, 
            Arnold DB
  TITLE     An E3-ligase-based method for ablating inhibitory synapses.
  JOURNAL   Nat Methods. 2016 Jun 6. doi: 10.1038/nmeth.3894.
  PUBMED    27271196
REFERENCE   2  (bases 1 to 7157)
  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: "Nat 
            Methods. 2016 Jun 6. doi: 10.1038/nmeth.3894."
COMMENT     Sequence Label: pCAG_EGFP-RandE3
FEATURES             Location/Qualifiers
     source          1..7157
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1670
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1594..1616
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     CDS             join(1742..1744,1745..1747,1748..1750,1751..2458)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MGVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT
                     LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKD
                     DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGI
                     KVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVL
                     LEFVTAAGITLGMDELY"
     CDS             join(1742..1744,1745..1747,1748..1750,1751..2458)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MGVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT
                     LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKD
                     DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGI
                     KVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVL
                     LEFVTAAGITLGMDELY"
     primer_bind     complement(1790..1811)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2051..2070)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2398..2419
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2780..2860
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     intron          3080..3213
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     3196..3215
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    3222..3810
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3275..3295)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3693..3704)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3837..3854)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3843..3954
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4246..4301
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4300..4319)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4660..4676)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4673..4695)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4684..4700
                     /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(4708..4714,4715..4732,4733..4738))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4753..4774
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4833..5028
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4879..5014
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5034..5168
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5071..5090)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5125..5144
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5236..5253)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5407..5995)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5487..5506)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6166..6957,6958..7026))
                     /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     6789..6808
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7027..7131)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg ggcgagggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct
     1321 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg
     1381 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc
     1441 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt
     1501 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg
     1561 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg
     1621 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca
     1681 acgtgctggt tgttgtgctg tctcatcatt ttggcaaaga attcggtacc gcgggcccgg
     1741 gatgggggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
     1801 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
     1861 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     1921 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
     1981 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     2041 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     2101 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     2161 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     2221 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     2281 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     2341 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     2401 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacgg
     2461 tggaggttct ggtggaggtt ctggaggtgg atcaggagga ggttcgcaat tgctcgaagt
     2521 cgtcgttgcg actccgacca gcctcctaat cagctggcgg gcgacgcagg agcacgcgcg
     2581 ctactaccgc atcacctacg gtgaaactgg tggcaatagc cctgtccagg aattcaccgt
     2641 gcctggcagc aagtccactg ctaccatcag cggcctgaaa cctggtgtcg actataccat
     2701 cacggtgtac gccgtcacgc gcgagcccca ggccagctcc gaccagtccc cgatctccat
     2761 caactaccgc accagatctt acccatacga tgttccagat tacgcttacc catacgatgt
     2821 tccagattac gcttacccat acgatgttcc agattacgct gctagcctaa ggcgcctaca
     2881 agaggagaag ctttgcaaaa tctgtatgga tagaaatatt gctatagttt ttgttccttg
     2941 tggacatctg gtcacttgta aacagtgtgc ggaagcagtt gacaaatgtc ccatgtgctg
     3001 cacagtcatt acgttcaagc aaaaaatttt tatgtcttaa taatgtacaa gtaaagcggc
     3061 cccggactcg aggccgcagg taagtatcaa ggttacaaga caggtttaag gagaccacat
     3121 agaaactggg cttgtcgaga cagagaagac tcttgcgttt ctgataggca cctattggtc
     3181 ttactgacat ccactttgcc tttctctcca caggtgtcga caatcaacct ctggattaca
     3241 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat
     3301 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
     3361 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
     3421 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca
     3481 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca
     3541 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
     3601 tggtgttgtc ggggaagctg acgtcctttc catggctgct cgcctgtgtt gccacctgga
     3661 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
     3721 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga
     3781 gtcggatctc cctttgggcc gcctccccgc ctggaattcg agctcggtac gatcagcctc
     3841 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     3901 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     3961 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     4021 ttgggaagac aatagcaggc cagcttttgt tccctttagg gttaattgcg cgcttggcgc
     4081 taatcatggt catagctgtt tccactcctc aggtgcaggc tgcctatcag aaggtggtgg
     4141 ctggtgtggc caatgccctg gctcacaaat accactgaga tcgttttccc tctgccaaaa
     4201 attatgggga catcatgaag ccccttgagc atctgacttc tggctaataa aggaaattta
     4261 ttttcattgc aatagtgtgt tggaattttt tgtgtctctc actcggaagg acatatggga
     4321 gggcaaatca tttaaaacat cagaatgagt atttggttta gagtttggca acatatgcca
     4381 tatgctggct gccatgaaca aaggtggcta taaagaggtc atcagtatat gaaacagccc
     4441 cctgctgtcc attccttatt ccatagaaaa gccttgactt gaggttagat ttttttttat
     4501 attttgtttg tgttattttt ttctttaaca tccctaaaat tttccttaca tgttttacta
     4561 gccagatttt tcctcctctc ctgactactc ccagtcatag ctgtccctct tctcttatga
     4621 agatccctcg acctgcagcc caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     4681 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     4741 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     4801 ccagtcggga aacctgtcgt gccagcggat ccgcatctca attagtcagc aaccatagtc
     4861 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     4921 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     4981 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctaacttgt
     5041 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     5101 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     5161 tctggatccg ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     5221 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     5281 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     5341 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     5401 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     5461 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     5521 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     5581 gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     5641 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     5701 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     5761 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     5821 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
     5881 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     5941 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     6001 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     6061 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     6121 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     6181 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     6241 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     6301 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     6361 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     6421 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     6481 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     6541 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     6601 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     6661 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     6721 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     6781 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     6841 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     6901 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     6961 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     7021 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     7081 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     7141 ccgaaaagtg ccacctg
//
