LOCUS       Exported                6109 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses channel rhodopsin in astrocytes.
ACCESSION   .
VERSION     .
KEYWORDS    pZac2.1 GfaABC1D ChR2(H134R) mCherry SV40
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6109)
  AUTHORS   Octeau JC, Gangwani MR, Allam SL, Tran D, Huang S, Hoang-Trong TM, 
            Golshani P, Rumbell TH, Kozloski JR, Khakh BS
  TITLE     Transient, Consequential Increases in Extracellular Potassium Ions 
            Accompany Channelrhodopsin2 Excitation.
  JOURNAL   Cell Rep. 2019 May 21;27(8):2249-2261.e7. doi: 
            10.1016/j.celrep.2019.04.078.
  PUBMED    31116972
REFERENCE   2  (bases 1 to 6109)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6109
                     /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          1239..1371
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1354..1373
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1416..1434
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1442..1451
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1448..2374
                     /codon_start=1
                     /product="H134R mutant of an N-terminal fragment of 
                     channelrhodopsin-2 from the alga Chlamydomonas reinhardtii 
                     (Nagel et al., 2005)"
                     /label=ChR2(H134R)
                     /note="blue light-activated cation channel"
                     /translation="MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGT
                     NGAQTASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKN
                     PSMLYLATGHRVQWLRYAEWLLTCPVILIRLSNLTGLSNDYSRRTMGLLVSDIGTIVWG
                     ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
                     WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
                     RKTTKLNIGGTEIEVETLVEDEAEAGAV"
     CDS             2387..3094
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(2529..2547)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2797..2816
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3134..3255
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3145..3164)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3199..3218
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3417..3546
                     /label=AAV2 ITR
     primer_bind     complement(3565..3582)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3565..3581)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3574..3596)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3723..4178
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3810..3829)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4020..4041
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4204..4308
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4309..5169
                     /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(4527..4546)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5340..5928
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5829..5848
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6082..6099
                     /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 gccagattta
      241 attaaggcct taattaggct gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc
      301 gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga
      361 gtggccaact ccatcactag gggttccttg tagttaatga ttaacccgcc atgctactta
      421 tctacgtagc catgctctag gaagatctaa catatcctgg tgtggagtag gggacgctgc
      481 tctgacagag gctcgggggc ctgagctggc tctgtgagct ggggaggagg cagacagcca
      541 ggccttgtct gcaagcagac ctggcagcat tgggctggcc gccccccagg gcctcctctt
      601 catgcccagt gaatgactca ccttggcaca gacacaatgt tcggggtggg cacagtgcct
      661 gcttcccgcc gcaccccagc ccccctcaaa tgccttccga gaagcccatt gagcaggggg
      721 cttgcattgc accccagcct gacagcctgg catcttggga taaaagcagc acagccccct
      781 aggggctgcc cttgctgtgt ggcgccaccg gcggtggaga acaaggctct attcagcctg
      841 tgcccaggaa aggggatcag gggatgccca ggcatggaca gtgggtggca gggggggaga
      901 ggagggctgt ctgcttccca gaagtccaag gacacaaatg ggtgagggga gagctctccc
      961 catagctggg ctgcggccca accccacccc ctcaggctat gccagggggt gttgccaggg
     1021 gcacccgggc atcgccagtc tagcccactc cttcataaag ccctcgcatc ccaggagcga
     1081 gcagagccag agcaggttgg agaggagacg catcacctcc gctgctcgca agctttattg
     1141 cggtagttta tcacagttaa attgctaacg cagtcagtgc ttctgacaca acagtctcga
     1201 acttaagctg cagaagttgg tcgtgaggca ctgggcaggt aagtatcaag gttacaagac
     1261 aggtttaagg agaccaatag aaactgggct tgtcgagaca gagaagactc ttgcgtttct
     1321 gataggcacc tattggtctt actgacatcc actttgcctt tctctccaca ggtgtccact
     1381 cccagttcaa ttacagctct taaggctaga gtacttaata cgactcacta taggctagcc
     1441 cgccaccatg gactatggcg gcgctttgtc tgccgtcgga cgcgaacttt tgttcgttac
     1501 taatcctgtg gtggtgaacg ggtccgtcct ggtccctgag gatcaatgtt actgtgccgg
     1561 atggattgaa tctcgcggca cgaacggcgc tcagaccgcg tcaaatgtcc tgcagtggct
     1621 tgcagcagga ttcagcattt tgctgctgat gttctatgcc taccaaacct ggaaatctac
     1681 atgcggctgg gaggagatct atgtgtgcgc cattgaaatg gttaaggtga ttctcgagtt
     1741 cttttttgag tttaagaatc cctctatgct ctaccttgcc acaggacacc gggtgcagtg
     1801 gctgcgctat gcagagtggc tgctcacttg tcctgtcatc cttatccgcc tgagcaacct
     1861 caccggcctg agcaacgact acagcaggag aaccatggga ctccttgtct cagacatcgg
     1921 gactatcgtg tggggggcta ccagcgccat ggcaaccggc tatgttaaag tcatcttctt
     1981 ttgtcttgga ttgtgctatg gcgcgaacac attttttcac gccgccaaag catatatcga
     2041 gggttatcat actgtgccaa agggtcggtg ccgccaggtc gtgaccggca tggcatggct
     2101 gtttttcgtg agctggggta tgttcccaat tctcttcatt ttggggcccg aaggttttgg
     2161 cgtcctgagc gtctatggct ccaccgtagg tcacacgatt attgatctga tgagtaaaaa
     2221 ttgttggggg ttgttgggac actacctgcg cgtcctgatc cacgagcaca tattgattca
     2281 cggagatatc cgcaaaacca ccaaactgaa catcggcgga acggagatcg aggtcgagac
     2341 tctcgtcgaa gacgaagccg aggccggagc cgtgccagcg gccgccgtga gcaagggcga
     2401 ggaggataac atggccatca tcaaggagtt catgcgcttc aaggtgcaca tggagggctc
     2461 cgtgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac
     2521 ccagaccgcc aagctgaagg tgaccaaggg tggccccctg cccttcgcct gggacatcct
     2581 gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag caccccgccg acatccccga
     2641 ctacttgaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga
     2701 cggcggcgtg gtgaccgtga cccaggactc ctccctgcag gacggcgagt tcatctacaa
     2761 ggtgaagctg cgcggcacca acttcccctc cgacggcccc gtaatgcaga agaagaccat
     2821 gggctgggag gcctcctccg agcggatgta ccccgaggac ggcgccctga agggcgagat
     2881 caagcagagg ctgaagctga aggacggcgg ccactacgac gctgaggtca agaccaccta
     2941 caaggccaag aagcccgtgc agctgcccgg cgcctacaac gtcaacatca agttggacat
     3001 cacctcccac aacgaggact acaccatcgt ggaacagtac gaacgcgccg agggccgcca
     3061 ctccaccggc ggcatggacg agctgtacaa gtaatctaga gtcgacccgg gcggccgctt
     3121 cgagcagaca tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga
     3181 aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc
     3241 tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag
     3301 atgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtaaaat cgataaggat
     3361 cttcctagag catggctacg tagataagta gcatggcggg ttaatcatta actacaagga
     3421 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
     3481 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
     3541 gcgcagcctt aattaaccta attcactggc cgtcgtttta caacgtcgtg actgggaaaa
     3601 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     3661 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     3721 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     3781 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     3841 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     3901 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     3961 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     4021 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     4081 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     4141 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat
     4201 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     4261 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     4321 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     4381 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     4441 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     4501 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     4561 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     4621 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     4681 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     4741 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     4801 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     4861 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     4921 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     4981 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     5041 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     5101 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     5161 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     5221 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     5281 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     5341 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     5401 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     5461 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     5521 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     5581 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     5641 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     5701 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     5761 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     5821 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     5881 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     5941 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     6001 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     6061 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaag
//
