LOCUS       Exported                7498 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Chemogenetics expression plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    pCAG PSAM4 GlyR IRES EGFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7498)
  AUTHORS   Magnus CJ, Lee PH, Bonaventura J, Zemla R, Gomez JL, Ramirez MH, Hu 
            X, Galvan A, Basu J, Michaelides M, Sternson SM
  TITLE     Ultrapotent chemogenetics for research and potential clinical 
            applications.
  JOURNAL   Science. 2019 Mar 14. pii: science.aav5282. doi: 
            10.1126/science.aav5282.
  PUBMED    30872534
REFERENCE   2  (bases 1 to 7498)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7498
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     38..60
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     122..141
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     misc_feature    1549..2134
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1716..1733)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             2147..2863
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2189..2210)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2450..2469)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2797..2818
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     intron          2908..3040
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     3023..3042
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    3049..3637
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3102..3122)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3520..3531)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3664..3681)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3670..3781
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3877..3897)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3877..3895)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4008..4027)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4073..4128
                     /label=-beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4127..4146)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4489..4505)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4489..4505)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4502..4524)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4513..4529
                     /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(4537..4567)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4582..4603
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4662..4857
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4708..4843
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4770..4789
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    4863..4997
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4900..4919)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4954..4973
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5065..5082)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5236..5824)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5316..5335)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5995..6855)
                     /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     6618..6637
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6856..6960)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        6990..7369
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
       61 ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg
      121 ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattcgg taccgcgggc
      181 ccggcgcgcc agctggatat ctccggaccc gggctagcgc caccatgcgc tgttctccag
      241 gcggcgtgtg gctcgccctg gctgcttccc ttctgcacgt tagcctgcag ggtgagttcc
      301 agcgcaaact gtataaggag cttgttaaga attataaccc cctggagcgg ccggtcgcaa
      361 atgattccca gccactgaca gtgtacttca gcctctcctt gctgcagatc atggacgtgg
      421 atgaaaagaa ccaggtgctg accactaata tttggttgca gatgtcctgg accgatcact
      481 acttgcagtg gaatgtgagc gaatacccag gtgtaaagac tgtaagattc cctgacggcc
      541 aaatctggaa accagatatc ctgctgtaca acagcgcaga cgaaaggttt gatgcaacat
      601 ttcacaccaa cgtgggagtc aattcttcag gccactgcct gtacctgccc cctggaatct
      661 tcaagtcctc atgctatatc gacgtccgct ggtttccctt cgacgtccag cactgcaaac
      721 tcaaattcgg gagctggagc tacggcggat ggagcctgga tctgcaaatg caggaggctg
      781 acatctctgg ttacatcccg aatggggagt gggaccttgt gggaatcccc ggtaaaagaa
      841 gcgagcgatt ttatgaatgc tgcaaagagc ccttcccaga tgtcaccttc acagtgacca
      901 tgcggagacg catgggttat tatctgatcc aaatgtatat cccaagcttg cttatagtga
      961 ttttgtcatg gatctccttc tggattaata tggacgccgc tccagctagg gtcggactgg
     1021 gcatcaccac agtgctgaca atgactactc agagctcagg cagccgagcc agcttgccca
     1081 aggtttctta cgtgaaggcc atcgatatct ggatggctgt ctgccttctg tttgtcttca
     1141 gcgcactgct ggaatacgcc gctgtcaatt ttgtgtctcg acagcataaa gagctgttgc
     1201 ggttcagaag aaaacgacgc caccacaaag aggatgaggc aggagaagga cgcttcaact
     1261 ttagcgccta tggtatggga cctgcttgcc tccaggctaa agacggaatt tccgtgaagg
     1321 gagccaacaa tagcaacaca accaacccac cccctgctcc atctaagagc ccggaggaaa
     1381 tgcgcaaact ctttattcag agagcgaaaa agatcgacaa aatctcccgg atcggattcc
     1441 ccatggcttt cctgattttc aacatgtttt attggatcat ctacaagatt gtgcgaaggg
     1501 aggacgtaca caaccagtaa gcggccgcaa ttccccccgc cccccccccc cctctccctc
     1561 ccccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg cgtttgtcta
     1621 tatgttattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga aacctggccc
     1681 tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct
     1741 gttgaatgtc gtgaaggaag cagttcctct ggaagcttct tgaagacaaa caacgtctgt
     1801 agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct gcggccaaaa
     1861 gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg
     1921 gatagttgtg gaaagagtca aatggctctc ctaagcgtat tcaacaaggg gctgaaggat
     1981 gcccagaagg taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca
     2041 tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga accacgggga cgtggttttc
     2101 ctttgaaaaa cacgatgata atatggccac aaccatgggg gatccggtga gcaagggcga
     2161 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     2221 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
     2281 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac
     2341 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     2401 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa
     2461 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     2521 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta
     2581 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt
     2641 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa
     2701 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc
     2761 caagctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     2821 cgccgccggg atcactctcg gcatggacga gctgtacaag taatctagag tcgaggtacc
     2881 gatgaatagc taaggtcgag gccgcaggta agtatcaagg ttacaagaca ggtttaagga
     2941 gaccaataga aactgggctt gtcgagacag agaagactct tgcgtttctg ataggcacct
     3001 attggtctta ctgacatcca ctttgccttt ctctccacag gtgtcgacaa tcaacctctg
     3061 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     3121 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
     3181 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
     3241 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
     3301 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
     3361 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     3421 aattccgtgg tgttgtcggg gaagctgacg tcctttccat ggctgctcgc ctgtgttgcc
     3481 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
     3541 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct
     3601 cagacgagtc ggatctccct ttgggccgcc tccccgcctg gaattcgagc tcggtacgat
     3661 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     3721 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     3781 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     3841 gggaggattg ggaagacaat agcccagctt ttgttccctt tagtgagggt taattgcgcg
     3901 cttggcgtaa tcatggtcat agcckaattc actcctcagg tgcaggctgc ctatcagaag
     3961 gtggtggctg gtgtggccaa tgccctggct cacaaatacc actgagatct ttttccctct
     4021 gccaaaaatt atggggacat catgaagccc cttgagcatc tgacttctgg ctaataaagg
     4081 aaatttattt tcattgcaat agtgtgttgg aattttttgt gtctctcact cggaaggaca
     4141 tatgggaggg caaatcattt aaaacatcag aatgagtatt tggtttagag tttggcaaca
     4201 tatgcccata tgctggctgc catgaacaaa ggttggctat aaagaggtca tcagtatatg
     4261 aaacagcccc ctgctgtcca ttccttattc catagaaaag ccttgacttg aggttagatt
     4321 ttttttatat tttgttttgt gttatttttt tctttaacat ccctaaaatt ttccttacat
     4381 gttttactag ccagattttt cctcctctcc tgactactcc cagtcatagc tgtccctctt
     4441 ctcttatgga gatccctcga cctgcagccc aagcttggcg taatcatggt catagctgtt
     4501 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     4561 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     4621 gcccgctttc cagtcgggaa acctgtcgtg ccagcggatc cgcatctcaa ttagtcagca
     4681 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     4741 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     4801 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     4861 ctaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4921 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     4981 cttatcatgt ctggatccgc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     5041 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     5101 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     5161 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     5221 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     5281 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     5341 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     5401 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     5461 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5521 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5581 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     5641 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     5701 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     5761 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     5821 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     5881 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     5941 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     6001 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     6061 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     6121 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     6181 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     6241 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     6301 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     6361 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     6421 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     6481 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     6541 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     6601 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     6661 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     6721 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     6781 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     6841 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     6901 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     6961 cacatttccc cgaaaagtgc cacctggtcg acattgatta ttgactagtt attaatagta
     7021 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
     7081 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
     7141 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
     7201 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
     7261 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
     7321 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tcgaggtgag
     7381 ccccacgttc tgcttcactc tccccatctc ccccccctcc ccacccccaa ttttgtattt
     7441 atttattttt taattatttt gtgcagcgat gggggcgggg gggggggggg cgcgcgcc
//
