LOCUS       Exported                7806 bp ds-DNA     circular SYN 24-MAY-2022
DEFINITION  Chemogenetics Cre-dependent expression plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    AAV SYN flex PSAM4 GlyR IRES EGFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7806)
  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 7806)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 24, 2022 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7806
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        143..173
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   259..388
                     /label=AAV2 ITR
     intron          987..1119
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1102..1121
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     protein_bind    complement(1140..1173)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     protein_bind    complement(1236..1269)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             complement(1282..1998)
                     /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(1327..1348)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1676..1695
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1935..1956
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     misc_feature    2011..2596
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     2412..2429
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     protein_bind    3961..3994
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     protein_bind    4065..4098
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    4166..4754
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4219..4239)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4637..4648)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4831..4952
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4842..4861)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4896..4915
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   5114..5243
                     /label=AAV2 ITR
     primer_bind     complement(5262..5279)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5262..5278)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5271..5293)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5420..5875
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5507..5526)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5717..5738
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5901..6005
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6006..6866
                     /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     complement(6224..6243)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7037..7625
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7526..7545
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7779..7796
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccargytca
      241 attaagscct tamtyargct gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc
      301 gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga
      361 gtggccaact ccatcactag gggttccttg tagttaatga ttaacccgcc atgctactta
      421 tctacgtagc catgctctag gaagatccga gctccagtgt gctggaattg gcccttttca
      481 gtatttaaag catgcttgac gaccgacccc gacccactgg acaagcaccc aacccccatt
      541 ccccaaattg cgcatcccct atcagagagg gggaggggaa acaggatgcg gcgaggcgcg
      601 tgcgcactgc cagcttcagc accgcggaca gtgccttcgc ccccgcctgg cggcgcgcgc
      661 caccgccgcc tcagcactga aggcgcgctg acgtcactcg ccggtccccc gcaaactccc
      721 cttcccggcc accttggtcg cgtccgcgcc gccgccggcc cagccggacc gcaccacgcg
      781 aggcgcgaga taggggggca cgggcgcgac catctgcgct gcggcgccgg cgactcagcg
      841 ctgcctcagt ctgcggtggg cagcggagga gtcgtgtcgt gcctgagagc gcagctgtgc
      901 tcctgggcac cgcgcagtcc gcccccgcgg ctcctggcca gaccacccct aggaccccct
      961 gccccaagtc gcagccgttg gatcaggtaa gtatcaaggt tacaagacag gtttaaggag
     1021 accaatagaa actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta
     1081 ttggtcttac tgacatccac tttgcctttc tctccacagg tgatgatggc gcgcccccca
     1141 taacttcgta taaagtatcc tatacgaagt tatatcaaaa taggaagacc aatgcttcac
     1201 catcgacccg aattgccaag catcaccatc gacacataac ttcgtataat gtatgctata
     1261 cgaagttata agcttgctag attacttgta cagctcgtcc atgccgagag tgatcccggc
     1321 ggcggtcacg aactccagca ggaccatgtg atcgcgcttc tcgttggggt ctttgctcag
     1381 cttggactgg gtgctcaggt agtggttgtc gggcagcagc acggggccgt cgccgatggg
     1441 ggtgttctgc tggtagtggt cggcgagctg cacgctgccg tcctcgatgt tgtggcggat
     1501 cttgaagttc accttgatgc cgttcttctg cttgtcggcc atgatataga cgttgtggct
     1561 gttgtagttg tactccagct tgtgccccag gatgttgccg tcctccttga agtcgatgcc
     1621 cttcagctcg atgcggttca ccagggtgtc gccctcgaac ttcacctcgg cgcgggtctt
     1681 gtagttgccg tcgtccttga agaagatggt gcgctcctgg acgtagcctt cgggcatggc
     1741 ggacttgaag aagtcgtgct gcttcatgtg gtcggggtag cggctgaagc actgcacgcc
     1801 gtaggtcagg gtggtcacga gggtgggcca gggcacgggc agcttgccgg tggtgcagat
     1861 gaacttcagg gtcagcttgc cgtaggtggc atcgccctcg ccctcgccgg acacgctgaa
     1921 cttgtggccg tttacgtcgc cgtccagctc gaccaggatg ggcaccaccc cggtgaacag
     1981 ctcctcgccc ttgctcaccg gatcccccat ggttgtggcc atattatcat cgtgtttttc
     2041 aaaggaaaac cacgtccccg tggttcgggg ggcctagacg tttttttaac ctcgactaaa
     2101 cacatgtaaa gcatgtgcac cgaggcccca gatcagatcc catacaatgg ggtaccttct
     2161 gggcatcctt cagccccttg ttgaatacgc ttaggagagc catttgactc tttccacaac
     2221 tatccaactc acaacgtggc actggggttg tgccgccttt gcaggtgtat cttatacacg
     2281 tggcttttgg ccgcagaggc acctgtcgcc aggtgggggg ttccgctgcc tgcaaagggt
     2341 cgctacagac gttgtttgtc ttcaagaagc ttccagagga actgcttcct tcacgacatt
     2401 caacagacct tgcattcctt tggcgagagg ggaaagaccc ctaggaatgc tcgtcaagaa
     2461 gacagggcca ggtttccggg ccctcacatt gccaaaagac ggcaatatgg tggaaaataa
     2521 catatagaca aacgcacacc ggccttattc caagcggctt cggccagtaa cgttaggggg
     2581 gggggaggga gagggggggg gggggggggn ggggggaatt gcggccgctt actggttgtg
     2641 tacgtcctcc cttcgcacaa tcttgtagat gatccaataa aacatgttga aaatcaggaa
     2701 agccatgggg aatccgatcc gggagatttt gtcgatcttt ttcgctctct gaataaagag
     2761 tttgcgcatt tcctccgggc tcttagatgg agcagggggt gggttggttg tgttgctatt
     2821 gttggctccc ttcacggaaa ttccgtcttt agcctggagg caagcaggtc ccataccata
     2881 ggcgctaaag ttgaagcgtc cttctcctgc ctcatcctct ttgtggtggc gtcgttttct
     2941 tctgaaccgc aacagctctt tatgctgtcg agacacaaaa ttgacagcgg cgtattccag
     3001 cagtgcgctg aagacaaaca gaaggcagac agccatccag atatcgatgg ccttcacgta
     3061 agaaaccttg ggcaagctgg ctcggctgcc tgagctctga gtagtcattg tcagcactgt
     3121 ggtgatgccc agtccgaccc tagctggagc ggcgtccata ttaatccaga aggagatcca
     3181 tgacaaaatc actataagca agcttgggat atacatttgg atcagataat aacccatgcg
     3241 tctccgcatg gtcactgtga aggtgacatc tgggaagggc tctttgcagc attcataaaa
     3301 tcgctcgctt cttttaccgg ggattcccac aaggtcccac tccccattcg ggatgtaacc
     3361 agagatgtca gcctcctgca tttgcagatc caggctccat ccgccgtagc tccagctccc
     3421 gaatttgagt ttgcagtgct ggacgtcgaa gggaaaccag cggacgtcga tatagcatga
     3481 ggacttgaag attccagggg gcaggtacag gcagtggcct gaagaattga ctcccacgtt
     3541 ggtgtgaaat gttgcatcaa acctttcgtc tgcgctgttg tacagcagga tatctggttt
     3601 ccagatttgg ccgtcaggga atcttacagt ctttacacct gggtattcgc tcacattcca
     3661 ctgcaagtag tgatcggtcc aggacatctg caaccaaata ttagtggtca gcacctggtt
     3721 cttttcatcc acgtccatga tctgcagcaa ggagaggctg aagtacactg tcagtggctg
     3781 ggaatcattt gcgaccggcc gctccagggg gttataattc ttaacaagct ccttatacag
     3841 tttgcgctgg aactcaccct gcaggctaac gtgcagaagg gaagcagcca gggcgagcca
     3901 cacgccgcct ggagaacagc gcatggtggc gctagcgaat tcggatccca tatgactagt
     3961 ataacttcgt ataggatact ttatacgaag ttatcattgg gattcttcct attttgatcc
     4021 aagcatcacc atcgaccctc tagtccagat ctcaccatcg acccataact tcgtatagca
     4081 tacattatac gaagttatgt ccctcgaaga ggttcgaaac actgaaaagg gcgaatttgc
     4141 agatgatcca ctcgaagctc ttaagaatca acctctggat tacaaaattt gtgaaagatt
     4201 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     4261 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     4321 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac
     4381 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc
     4441 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc
     4501 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa
     4561 atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc
     4621 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc
     4681 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg
     4741 ggccgcctcc ccgcatcgat accgtcgacc cgggcggccg tcgaatgcat ctaggtcgcc
     4801 cgggcggccg gccgcttcga gcagacatga taagatacat tgatgagttt ggacaaacca
     4861 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
     4921 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt
     4981 ttcaggttca gggggagatg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg
     5041 gtaaaatcga taaggatctt cctagagcat ggctacgtag ataagtagca tggcgggtta
     5101 atcattaact acaaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc
     5161 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc
     5221 tcagtgagcg agcgagcgcg cagccttaat taacctaatt cactggccgt cgttttacaa
     5281 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     5341 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     5401 agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     5461 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     5521 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     5581 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     5641 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     5701 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     5761 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     5821 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
     5881 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     5941 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     6001 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     6061 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     6121 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     6181 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     6241 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     6301 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     6361 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     6421 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     6481 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     6541 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     6601 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     6661 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     6721 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     6781 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     6841 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     6901 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     6961 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     7021 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     7081 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     7141 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     7201 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     7261 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     7321 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     7381 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     7441 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     7501 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     7561 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     7621 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     7681 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     7741 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     7801 cggaag
//
