LOCUS       Exported                8371 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  pAAV vector to express H2B-GFP and oG (optimized Glycoprotein) in a 
            Cre-dependent manner.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-Ef1a-DIO-H2B-GFP-2A-oG-WPRE-hGH
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8371)
  AUTHORS   Kim EJ, Jacobs MW, Ito-Cole T, Callaway EM
  TITLE     Improved Monosynaptic Neural Circuit Tracing Using Engineered Rabies
            Virus Glycoproteins
  JOURNAL   Cell Reports 2016
REFERENCE   2  (bases 1 to 8371)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8371
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        242..1419
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          473..1410
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1367..1387
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    1450..1483
                     /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    1534..1567
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             complement(3151..3216)
                     /codon_start=1
                     /product="2A peptide from foot-and-mouth disease virus 
                     polyprotein"
                     /label=F2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="VKQTLNFDLLKLAGDVESNPGP"
     CDS             complement(3226..3942)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(3226..3942)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3268..3289)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3617..3636
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3876..3897
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      3939..3948
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             complement(3961..4338)
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     protein_bind    complement(4356..4389)
                     /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(4440..4473)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    4498..5086
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4551..4571)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4969..4980)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    5118..5594
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(5317..5336)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   5634..5774
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5634..5763
                     /label=AAV2 ITR
     rep_origin      5849..6304
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5936..5955)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6146..6167
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6321..6340)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6440..6462
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6500..6518)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6586..6690
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6691..7551
                     /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(6909..6928)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7722..8310
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8211..8230
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtaagct ttgcaaagat ggataaagtt
      181 ttaaacagag aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat
      241 tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
      301 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
      361 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
      421 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc
      481 cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat
      541 tacttccact ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      601 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      661 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      721 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      781 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      841 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      901 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      961 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
     1021 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
     1081 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
     1141 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
     1201 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1261 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1321 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1381 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc
     1441 gactccggaa taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga
     1501 ttgtagctgc tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg
     1561 aagttatggc gcgccttaga gccgtgtctc gcccccagat ttatgacttt cccaactgct
     1621 gatgatttta ccgctctggg gtgtgacaga cacttctcta ccggtccccc tcagattgtg
     1681 ctgagttggc tcggagcggt tcactcgtcg ccagcaggtc atcaggaaaa tgatcagcat
     1741 gagagcagtc agtgctccag cggagagcag cacgtacttg ccccaattag gcagtccgag
     1801 atcgacgcca ctgatccgtt cgtgcacgtc gggcaggtgg acctccacaa aatcttcggc
     1861 ctcgtcgccg tttttgaaca ctgtgcttgg gtctgcgaga ggatgcatca ggggaatgac
     1921 gcttgacacg agcagttcca tgtgctgctg gagcagacta gactgcatct cggggatcag
     1981 cacgttacca tctggcccca gaatgatacc attaaagaag accccgttca catgtgggtg
     2041 acatcgaccc ccgactcgca ggcatccctt actaggaatg atttcattcc aggttctcac
     2101 agatttataa tgggcgtctg cctccatcag tgtcttgtta aagatggtgt atgcttttcc
     2161 gaagccaggg acgagcttcc tcaggtggct cagcctcctg aagctcacgc tcttggtagt
     2221 cataattgac tccagagcat cgagacattc ctcccttttc ttcacgagtt cctcgaccac
     2281 cagatgttcg atctcatcgc ttcggaagtc gtgcagattc acgagctgcc caggggggca
     2341 ccacttagtc tcgttagatg tctgcatagc gacccaggtt ccgtccatca gccggagtcc
     2401 cagcacgcca cagagtttca gcttgcaggc acctttgagg ctcttataca gccctctttc
     2461 atccacaaat ccacaagtct cgctgccttt tgaggccctc ttgcccctgc tgttggtgaa
     2521 gatgtcgcag gacatcccca gccttggatt ctcaggcatc cagattgtat aatcatggtt
     2581 ggtggaacag taagtggagc tgacggccac tccggagcag tttccgccgg gaaacactgg
     2641 actgtggaga gatcggtcgt aggggtccag gtcggcgaca gatggggaaa tgatgaccag
     2701 gctctctttt gtggtcttca cagtgcgcag ccaatgatag tctgggtaag gattgtgcag
     2761 gctttcctca tagcgggggt cgccagccat cttccagttg taagctgccc gacaggcgtc
     2821 tggtgttggc cgaaagtgtt ttctcttgaa agttgtggtg acatatccca caaaatttgt
     2881 gtaggtttct gcctcggtga ccacaccagt gcatgtgaac ccgttcatct taatagcaga
     2941 gatatagccc actttcagct ccatgtagga gaatccgctc aggttggtac agccttcgtc
     3001 ctcgaccacg agattgttag ggcatgacag atggtgaata tcgataggac tccaggggcc
     3061 cagcttgtcg gggatagtgt agatagggaa tttaccaaag cagagtggga agacgagcag
     3121 agggacgaag aggagagcct gtgggaccat gggccctggg ttggactcca cgtctcccgc
     3181 caacttgaga aggtcaaaat tcaaagtctg tttcacacca ctacccttgt acagctcgtc
     3241 catgccgaga gtgatcccgg cggcggtcac gaactccagc aggaccatgt gatcgcgctt
     3301 ctcgttgggg tctttgctca gggcggactg ggtgctcagg tagtggttgt cgggcagcag
     3361 cacggggccg tcgccgatgg gggtgttctg ctggtagtgg tcggcgagct gcacgctgcc
     3421 gtcctcgatg ttgtggcgga tcttgaagtt caccttgatg ccgttcttct gcttgtcggc
     3481 catgatatag acgttgtggc tgttgtagtt gtactccagc ttgtgcccca ggatgttgcc
     3541 gtcctccttg aagtcgatgc ccttcagctc gatgcggttc accagggtgt cgccctcgaa
     3601 cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg tgcgctcctg
     3661 gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt ggtcggggta
     3721 gcggctgaag cactgcacgc cgtaggtcag ggtggtcacg agggtgggcc agggcacggg
     3781 cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg ccgtaggtgg catcgccctc
     3841 gccctcgccg gacacgctga acttgtggcc gtttacgtcg ccgtccagct cgaccaggat
     3901 gggcaccacc ccggtgaaca gctcctcgcc cttgctcacc atggtggcga ccggtggatc
     3961 cttagcgctg gtgtacttgg tgatggcctt agtaccctcg gacacggcgt gcttggccaa
     4021 ctccccaggc agcagcaggc gcacggccgt ctggatctcc ctggaggtga tggtcgagcg
     4081 cttgttgtaa tgcgccaggc gggaagcctc acctgcgatg cgctcgaaaa tgtcgttcac
     4141 aaacgaattc atgatgccca tggccttgga cgaaatgccg gtgtcagggt ggacctgctt
     4201 cagaaccttg tacacataga tggaatagct ctccttgcgg ctgcgcttgc gcttcttgcc
     4261 gcctttcttc tgcgccttag tcaccgcctt cttggagccc tttttcgggg cgggagcaga
     4321 cttcgctggc tctggcatgg tggcggcggg ctagcataac ttcgtataaa gtatcctata
     4381 cgaagttatt tgccttaacc cagaaattat cactgttatt ctttagaatg gtgcaaagaa
     4441 taacttcgta taatgtatgc tatacgaagt tatgaattcg atatcaagct tatcgataat
     4501 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     4561 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     4621 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     4681 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     4741 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     4801 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     4861 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
     4921 tatgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     4981 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     5041 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgagcg
     5101 ctgctcgaga gatctacggg tggcatccct gtgacccctc cccagtgcct ctcctggccc
     5161 tggaagttgc cactccagtg cccaccagcc ttgtcctaat aaaattaagt tgcatcattt
     5221 tgtctgacta ggtgtccttc tataatatta tggggtggag gggggtggta tggagcaagg
     5281 ggcaagttgg gaagacaacc tgtagggcct gcggggtcta ttgggaacca agctggagtg
     5341 cagtggcaca atcttggctc actgcaatct ccgcctcctg ggttcaagcg attctcctgc
     5401 ctcagcctcc cgagttgttg ggattccagg catgcatgac caggctcagc taatttttgt
     5461 ttttttggta gagacggggt ttcaccatat tggccaggct ggtctccaac tcctaatctc
     5521 aggtgatcta cccaccttgg cctcccaaat tgctgggatt acaggcgtga accactgctc
     5581 ccttccctgt ccttctgatt ttgtaggtaa ccacgtgcgg accgagcggc cgcaggaacc
     5641 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     5701 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     5761 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     5821 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     5881 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     5941 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     6001 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     6061 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     6121 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     6181 cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     6241 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     6301 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     6361 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     6421 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     6481 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     6541 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     6601 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     6661 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     6721 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     6781 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     6841 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     6901 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     6961 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     7021 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     7081 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     7141 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     7201 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     7261 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     7321 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     7381 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     7441 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     7501 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     7561 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     7621 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     7681 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     7741 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     7801 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     7861 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     7921 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     7981 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     8041 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     8101 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     8161 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     8221 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     8281 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     8341 ctggcctttt gctggccttt tgctcacatg t
//
