LOCUS       Exported                7772 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Express cre dependent TVA and SADB19 RG (biscistronic) as an AAV.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-EF1a-FLEX-T-RG
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7772)
  AUTHORS   Velez-Fort M, Bracey EF, Keshavarzi S, Rousseau CV, Cossell L, Lenzi
            SC, Strom M, Margrie TW
  TITLE     A Circuit for Integration of Head- and Visual-Motion Signals in 
            Layer 6 of Mouse Primary Visual Cortex.
  JOURNAL   Neuron. 2018 Apr 4;98(1):179-191.e6. doi: 
            10.1016/j.neuron.2018.02.023. Epub 2018 Mar 15.
  PUBMED    29551490
REFERENCE   2  (bases 1 to 7772)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7772
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      211..666
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(298..317)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     508..529
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(683..702)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     802..824
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(862..880)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        948..1052
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1053..1913
                     /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(1271..1290)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2084..2672
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2573..2592
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2734..2863
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        2964..4142
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3195..4133
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4090..4110
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      4164..4173
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    complement(4176..4209)
                     /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(4272..4305)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             complement(5896..5955)
                     /codon_start=1
                     /product="2A peptide from equine rhinitis A virus 
                     polyprotein"
                     /label=E2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="QCTNYALLKLAGDVESNPGP"
     protein_bind    6453..6486
                     /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    6557..6590
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    6632..7220
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6685..6705)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7103..7114)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    7252..7728
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(7451..7470)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   7768..136
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7768..125
                     /label=AAV2 ITR
ORIGIN
        1 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg
       61 cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg
      121 cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
      181 tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg
      241 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc
      301 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
      361 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
      421 ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
      481 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
      541 actctatctc gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt
      601 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
      661 caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
      721 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
      781 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
      841 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
      901 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
      961 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     1021 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     1081 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     1141 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     1201 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     1261 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     1321 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     1381 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     1441 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     1501 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     1561 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     1621 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     1681 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     1741 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     1801 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     1861 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     1921 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     1981 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     2041 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     2101 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     2161 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     2221 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     2281 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     2341 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     2401 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     2461 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     2521 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     2581 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     2641 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     2701 tcctggcctt ttgctggcct tttgctcaca tgtcctgcag gcagctgcgc gctcgctcgc
     2761 tcactgaggc cgcccgggcg tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga
     2821 gcgcgcagag agggagtggc caactccatc actaggggtt cctgcggccg cacgcgtaag
     2881 ctttgcaaag atggataaag ttttaaacag agaggaatct ttgcagctaa tggaccttct
     2941 aggtcttgaa aggagtggga attggctccg gtgcccgtca gtgggcagag cgcacatcgc
     3001 ccacagtccc cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaggt
     3061 ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg
     3121 ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg
     3181 ccgccagaac acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt
     3241 atggcccttg cgtgccttga attacttcca cctggctgca gtacgtgatt cttgatcccg
     3301 agcttcgggt tggaagtggg tgggagagtt cgaggccttg cgcttaagga gccccttcgc
     3361 ctcgtgcttg agttgaggcc tggcctgggc gctggggccg ccgcgtgcga atctggtggc
     3421 accttcgcgc ctgtctcgct gctttcgata agtctctagc catttaaaat ttttgatgac
     3481 ctgctgcgac gctttttttc tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca
     3541 ctggtatttc ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat
     3601 gttcggcgag gcggggcctg cgagcgcggc caccgagaat cggacggggg tagtctcaag
     3661 ctggccggcc tgctctggtg cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg
     3721 caaggctggc ccggtcggca ccagttgcgt gagcggaaag atggccgctt cccggccctg
     3781 ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt gagtcaccca
     3841 cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc
     3901 gggcgccgtc caggcacctc gattagttct cgagcttttg gagtacgtcg tctttaggtt
     3961 ggggggaggg gttttatgcg atggagtttc cccacactga gtgggtggag actgaagtta
     4021 ggccagcttg gcacttgatg taattctcct tggaatttgc cctttttgag tttggatctt
     4081 ggttcattct caagcctcag acagtggttc aaagtttttt tcttccattt caggtgtcgt
     4141 gaggtaccgg atcctctaga gccgccacca tggccataac ttcgtataaa gtatcctata
     4201 cgaagttata tcaaaatagg aagaccaatg cttcaccatc gacccgaatt gccaagcatc
     4261 accatcgacc cataacttcg tataatgtat gctatacgaa gttatacttc aactagttca
     4321 cagtctggtc tcgccgccgc tcttgtggct ctcccagctg ctgatgatct tgccgctctg
     4381 gggggtcacg ctcacctctc tgccggtgcc tctcaggttg tgctgggtgg gctcgcttct
     4441 gttcactctt ctgcagcagg tcatcaggaa gatgatcagc atcagggcgg tcagggcgcc
     4501 ggcgctcagc agcacgtact tgccccagtt gggcaggccc aggtccacgc cgctcacctg
     4561 gttgtgcacg tcgggcaggt gcacctccac gaagtcctcg gcctcgtcgc cgtccttgaa
     4621 cacggtgctg gggtcggcca gggggtgcac cagggggatc acgctgctct ccagcagctc
     4681 catgtgctgc tgcagcaggc tgctctgcat ctcggggatc agcacgttgc cgtcggggcc
     4741 caggatgatg ccgttgaaga acacgccgtt cacgtggggg tggcatctgc cgcccactct
     4801 caggcagccc ttgctgggca ggatctcgtt ccaggttctc acgctcttgt agtgggcgtc
     4861 ggcctccatc agggtcttgt tgaagatggt gtaggccttg ccgaagccgg gcaccagctt
     4921 tctcaggtgg ctcagtcttc tgaagctcac gctcttggtg gtcatgatgc tctccagggc
     4981 gtccaggcac tcctctctct ttctcaccag ctcctccacc accaggtgct cgatctcgtc
     5041 gcttctgaag tcgtgcaggt tcaccagctt gtcggggggg caccacttgg tctcgttgct
     5101 ggtctgcatg ctcacccagg tgccgtccat cagtctcagg cccagcacgc cgcacagctt
     5161 cagcttgcag gcgcccttca ggctcttgta caggcctctc tcgtccacga agccgcaggt
     5221 ctcgctgccc ttgctggctc tcttgcctct gctgttggtg aagatgtcgc agctcatgcc
     5281 cagtctgggg ttctcgggca tccagatggt gtagtcgtgg ttggtgctgc agtaggtgct
     5341 gctcacggcc acgccgctgc acttgccgct ggggaacact ctgctgtgca ggcttctgtc
     5401 gtaggggtcc aggtcggcca cgctggggct gatgatcacc aggctctcct tggtggtctt
     5461 cacggttctc agccatctgt agtcggggta ggggttgtgc aggctctcct cgtatctggg
     5521 gtcgccggcc atcttccagt tgtaggcggc tctgcaggcg tcgggggtgg gtctgaagtg
     5581 ctttctcttg aaggtggtgg tcacgtagcc cacgaagttg gtgtaggtct cggcctcggt
     5641 caccacgccg gtgcaggtga agccgttcac cttgatggcc aggatgtagc ccaccttcag
     5701 ctccatgtag ctgaagccgc tcaggttggt gcagccctcg tcctccacca ccaggttgtt
     5761 ggggcagctc aggtggtgga tgtcgatggg gctccagggg cccagcttgt cggggatggt
     5821 gtagatgggg aacttgccga agcacagggg gaacaccagc aggggcacga acagcagggc
     5881 ctggggcact ccggaaggac cggggttact ttcaacatcg ccagcgagtt tcaacaaagc
     5941 gtagttagta cattgaccac taccgctagc ggagaacaag tctgcctggc tcttgtcagg
     6001 caccagcagc tccttggatg cgctttcaag actgaagatg tcagacctgc tttttgcttt
     6061 ggacttcccc catgcagcga taccacccac agctaccagg cagcacagga gcactgcagt
     6121 gatcagcatc cacatgcggc cgtgattcct ggctggcaga gcacgtccag gagcaggggc
     6181 agtgggagcc tctgtgccgt tgtccgtggg caccgcgggg atcgcgctgg tcccgcagcc
     6241 ccactcgtcc cgcccgtcgt cgcagtcggg gtgtccgtcg cacagccagt cctgcgggta
     6301 acactccccg tgggcaccgg gcggctccga gcagcggaac tgaccggggg ggcaacggga
     6361 caaagaaccg ttaccggacc cgttaccggt cacgttaccg ggcagcagca gcagcagcag
     6421 cgcgggcagc agccgcgcat cgatgtcgac gcataacttc gtataggata ctttatacga
     6481 agttatcttt gggattcttc ctattttgat ccaagcatca ccatcgaccc tctagtccag
     6541 atctcaccat cgacccataa cttcgtatag catacattat acgaagttat gtccctcgag
     6601 gaggttcgaa ttcgatatca agcttatcga taatcaacct ctggattaca aaatttgtga
     6661 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     6721 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     6781 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     6841 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     6901 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     6961 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
     7021 ggggaaatca tcgtcctttc cttggctgct cgcctatgtt gccacctgga ttctgcgcgg
     7081 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     7141 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     7201 cctttgggcc gcctccccgc atcgataccg agcgctgctc gagagatcta cgggtggcat
     7261 ccctgtgacc cctccccagt gcctctcctg gccctggaag ttgccactcc agtgcccacc
     7321 agccttgtcc taataaaatt aagttgcatc attttgtctg actaggtgtc cttctataat
     7381 attatggggt ggaggggggt ggtatggagc aaggggcaag ttgggaagac aacctgtagg
     7441 gcctgcgggg tctattggga accaagctgg agtgcagtgg cacaatcttg gctcactgca
     7501 atctccgcct cctgggttca agcgattctc ctgcctcagc ctcccgagtt gttgggattc
     7561 caggcatgca tgaccaggct cagctaattt ttgttttttt ggtagagacg gggtttcacc
     7621 atattggcca ggctggtctc caactcctaa tctcaggtga tctacccacc ttggcctccc
     7681 aaattgctgg gattacaggc gtgaaccact gctcccttcc ctgtccttct gattttgtag
     7741 gtaaccacgt gcggaccgag cggccgcagg aa
//
