LOCUS       Exported                7162 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses under the CAG promoter GFE3, a synthetic E3 ligase that 
            rapidly degrades endogenous Gephyrin at inhibitory synapses..
ACCESSION   .
VERSION     .
KEYWORDS    pCAG_EGFP-GFE3
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7162)
  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 7162)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7162
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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             1742..2458
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MGVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT
                     LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKD
                     DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGI
                     KVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVL
                     LEFVTAAGITLGMDELY"
     CDS             1742..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             2786..2866
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     intron          3086..3218
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     3201..3220
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    3227..3815
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3280..3300)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3698..3709)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3842..3859)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3848..3959
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4251..4306
                     /label=-beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4305..4324)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4665..4681)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4665..4681)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4678..4700)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4689..4705
                     /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(4713..4743)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4758..4779
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4838..5033
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4884..5019
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4946..4965
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    5039..5173
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5076..5095)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5130..5149
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5241..5258)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5412..6000)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5492..5511)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6171..7031)
                     /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     6794..6813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7032..7136)
                     /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 caaggaagca tcaccaacca gcatccagat cagctggggc aagtacaagg tcatggttcg
     2581 ctactaccgc atcacctacg gtgaaactgg tggcaatagc cctgtccagg aattcaccgt
     2641 gcctggcagc aagtccactg ctaccatcag cagcctgaaa cctggtgtcg actataccat
     2701 cacggtgtac gccgtcacga tcgaccactg gaactaccag gacccgatcc cgatctccat
     2761 caactaccgc accggttcaa gatcttaccc atacgatgtt ccagattacg cttacccata
     2821 cgatgttcca gattacgctt acccatacga tgttccagat tacgctgcta gcctaaggcg
     2881 cctacaagag gagaagcttt gcaaaatctg tatggataga aatattgcta tagtttttgt
     2941 tccttgtgga catctggtca cttgtaaaca gtgtgcggaa gcagttgaca aatgtcccat
     3001 gtgctgcaca gtcattacgt tcaagcaaaa aatttttatg tcttaataat gtacaagtaa
     3061 agcggccccg gactcgaggc cgcaggtaag tatcaaggtt acaagacagg tttaaggaga
     3121 ccaatagaaa ctgggcttgt cgagacagag aagactcttg cgtttctgat aggcacctat
     3181 tggtcttact gacatccact ttgcctttct ctccacaggt gtcgacaatc aacctctgga
     3241 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
     3301 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     3361 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
     3421 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
     3481 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
     3541 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     3601 ttccgtggtg ttgtcgggga agctgacgtc ctttccatgg ctgctcgcct gtgttgccac
     3661 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
     3721 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
     3781 gacgagtcgg atctcccttt gggccgcctc cccgcctgga attcgagctc ggtacgatca
     3841 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     3901 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     3961 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     4021 gaggattggg aagacaatag caggccagct tttgttccct ttagggttaa ttgcgcgctt
     4081 ggcgctaatc atggtcatag ctgtttccac tcctcaggtg caggctgcct atcagaaggt
     4141 ggtggctggt gtggccaatg ccctggctca caaataccac tgagatcgtt ttccctctgc
     4201 caaaaattat ggggacatca tgaagcccct tgagcatctg acttctggct aataaaggaa
     4261 atttattttc attgcaatag tgtgttggaa ttttttgtgt ctctcactcg gaaggacata
     4321 tgggagggca aatcatttaa aacatcagaa tgagtatttg gtttagagtt tggcaacata
     4381 tgccatatgc tggctgccat gaacaaaggt ggctataaag aggtcatcag tatatgaaac
     4441 agccccctgc tgtccattcc ttattccata gaaaagcctt gacttgaggt tagatttttt
     4501 tttatatttt gtttgtgtta tttttttctt taacatccct aaaattttcc ttacatgttt
     4561 tactagccag atttttcctc ctctcctgac tactcccagt catagctgtc cctcttctct
     4621 tatgaagatc cctcgacctg cagcccaagc ttggcgtaat catggtcata gctgtttcct
     4681 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     4741 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     4801 gctttccagt cgggaaacct gtcgtgccag cggatccgca tctcaattag tcagcaacca
     4861 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     4921 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
     4981 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctaa
     5041 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     5101 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     5161 tcatgtctgg atccgctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5221 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5281 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5341 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5401 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5461 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     5521 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     5581 ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag
     5641 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     5701 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     5761 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     5821 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg
     5881 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     5941 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     6001 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     6061 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     6121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     6181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     6241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     6301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     6361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     6421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     6481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     6541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     6601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     6661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     6781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     6841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     6901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     6961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     7021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     7081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     7141 tttccccgaa aagtgccacc tg
//
