LOCUS       Exported                6192 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  fusion protein of Gaussia luciferase variant slow burn, B7 
            transmembrane region, and enhanced yellow fluorescent protein; 
            control for bioluminescent optogenetics.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-hSyn-sbGLuc-B7-EYFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6192)
  AUTHORS   Gomez-Ramirez M, More AI, Friedman NG, Hochgeschwender U, Moore CI
  TITLE     The BioLuminescent-OptoGenetic in vivo response to coelenterazine is
            proportional, sensitive, and specific in neocortex.
  JOURNAL   J Neurosci Res. 2020 Mar;98(3):471-480. doi: 10.1002/jnr.24498. Epub
            2019 Sep 23.
  PUBMED    31544973
REFERENCE   2  (bases 1 to 6192)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6192
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   5..134
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        179..626
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      659..668
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             665..1219
                     /codon_start=1
                     /product="enhanced variant of secreted Gaussia luciferase"
                     /label=Gaussia-Dura Luc
                     /note="human codon-optimized"
                     /translation="MGVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTDLDADRG
                     KLPGKKLPLEVLKELEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESA
                     QGGIGEAIVDIPEIPGFKDLEPLEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQ
                     RCATFASKIQGQVDKIKGAGGD"
     CDS             665..1219
                     /codon_start=1
                     /product="secreted Gaussia luciferase"
                     /label=hGLuc
                     /note="human codon-optimized"
                     /translation="MGVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTDLDADRG
                     KLPGKKLPLEVLKELEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESA
                     QGGIGEAIVDIPEIPGFKDLEPLEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQ
                     RCATFASKIQGQVDKIKGAGGD"
     CDS             1478..2194
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1520..1541)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1781..1800)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2128..2149
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2224..2265
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2275..2292
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    2323..2911
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2376..2396)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2794..2805)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2943..3419
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3142..3161)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3459..3599
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3459..3588
                     /label=AAV2 ITR
     rep_origin      3674..4129
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3761..3780)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3971..3992
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4146..4165)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4265..4287
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4325..4343)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4411..4515
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4516..5376
                     /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(4734..4753)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5547..6135
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6036..6055
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atgtcctgca ggcagctgcg cgctcgctcg ctcactgagg ccgcccgggc gtcgggcgac
       61 ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat
      121 cactaggggt tcctgcggcc gcacgcgtgt gtctagactg cagagggccc tgcgtatgag
      181 tgcaagtggg ttttaggacc aggatgaggc ggggtggggg tgcctacctg acgaccgacc
      241 ccgacccact ggacaagcac ccaaccccca ttccccaaat tgcgcatccc ctatcagaga
      301 gggggagggg aaacaggatg cggcgaggcg cgtgcgcact gccagcttca gcaccgcgga
      361 cagtgccttc gcccccgcct ggcggcgcgc gccaccgccg cctcagcact gaaggcgcgc
      421 tgacgtcact cgccggtccc ccgcaaactc cccttcccgg ccaccttggt cgcgtccgcg
      481 ccgccgccgg cccagccgga ccgcaccacg cgaggcgcga gatagggggg cacgggcgcg
      541 accatctgcg ctgcggcgcc ggcgactcag cgctgcctca gtctgcggtg ggcagcggag
      601 gagtcgtgtc gtgcctgaga gcgcagtcga gaaggtaccg gatctcgagc tcaagcttgc
      661 caccatggga gtcaaagttc tgtttgccct gatctgcatc gctgtggccg aggccaagcc
      721 caccgagaac aacgaagact tcaacatcgt ggccgtggcc agcaacttcg cgaccacgga
      781 tctcgatgct gaccgcggga agttgcccgg caagaagctg ccgctggagg tgctcaaaga
      841 gctggaagcc aatgcccgga aagctggctg caccaggggc tgtctgatct gcctgtccca
      901 catcaagtgc acgcccaaga tgaagaagtt catcccagga cgctgccaca cctacgaagg
      961 cgacaaagag tccgcacagg gcggcatagg cgaggcgatc gtcgacattc ctgagattcc
     1021 tgggttcaag gacttggagc ccctggagca gttcatcgca caggtcgatc tgtgtgtgga
     1081 ctgcacaact ggctgcctca aagggcttgc caacgtgcag tgttctgacc tgctcaagaa
     1141 gtggctgccg caacgctgtg cgacctttgc cagcaagatc cagggccagg tggacaagat
     1201 caagggggcc ggtggtgacg gcgacccact agtccagtgt ggtggaattg ccggatcccc
     1261 cccagaagac cctcctgata gcaagaacac acttgtgctc tttggggcag gattcggcgc
     1321 agtaataaca gtcgtcgtca tcgttgtcat catcaaatgc ttctgtaagc acagaagctg
     1381 tttcagaaga aatgaggcaa gcagagaaac aaacaacagc cttaccttcg ggcctgaaga
     1441 agcattagct gaacagaccg tcttccttgc ggccgccgtg agcaagggcg aggagctgtt
     1501 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     1561 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     1621 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccttcg gctacggcct
     1681 gcagtgcttc gcccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     1741 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     1801 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     1861 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     1921 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     1981 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     2041 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agctaccagt ccgccctgag
     2101 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     2161 gatcactctc ggcatggacg agctgtacaa gtaacatatg ctctagaggg cccgcggttc
     2221 gaaggtaagc ctatccctaa ccctctcctc ggtctcgatt ctacgcgtac cggtcatcat
     2281 caccatcacc attgagttta attcgatatc aagcttatcg ataatcaacc tctggattac
     2341 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
     2401 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
     2461 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
     2521 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc
     2581 acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc
     2641 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
     2701 gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctatgt tgccacctgg
     2761 attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct
     2821 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg
     2881 agtcggatct ccctttgggc cgcctccccg catcgatacc gagcgctgct cgagagatct
     2941 acgggtggca tccctgtgac ccctccccag tgcctctcct ggccctggaa gttgccactc
     3001 cagtgcccac cagccttgtc ctaataaaat taagttgcat cattttgtct gactaggtgt
     3061 ccttctataa tattatgggg tggagggggg tggtatggag caaggggcaa gttgggaaga
     3121 caacctgtag ggcctgcggg gtctattggg aaccaagctg gagtgcagtg gcacaatctt
     3181 ggctcactgc aatctccgcc tcctgggttc aagcgattct cctgcctcag cctcccgagt
     3241 tgttgggatt ccaggcatgc atgaccaggc tcagctaatt tttgtttttt tggtagagac
     3301 ggggtttcac catattggcc aggctggtct ccaactccta atctcaggtg atctacccac
     3361 cttggcctcc caaattgctg ggattacagg cgtgaaccac tgctcccttc cctgtccttc
     3421 tgattttgta ggtaaccacg tgcggaccga gcggccgcag gaacccctag tgatggagtt
     3481 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg
     3541 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg
     3601 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca
     3661 aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3721 cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct
     3781 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     3841 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt
     3901 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     3961 ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat
     4021 tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt
     4081 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact
     4141 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     4201 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     4261 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     4321 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     4381 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     4441 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     4501 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     4561 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     4621 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     4681 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     4741 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     4801 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     4861 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     4921 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     4981 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     5041 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     5101 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     5161 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     5221 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     5281 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     5341 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     5401 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     5461 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     5521 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     5581 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     5641 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     5701 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     5761 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     5821 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     5881 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     5941 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     6001 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     6061 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     6121 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     6181 ccttttgctc ac
//
