LOCUS       Exported                7294 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Gateway destination transfer plasmid for making AAV. Contains a DIO 
            cassette for cre-induction. Without cre, insert is backwards in 
            reference to promoter..
ACCESSION   .
VERSION     .
KEYWORDS    pAAV_GW_Dest
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7294)
  TITLE     Mitra lab plasmids
REFERENCE   2  (bases 1 to 7294)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7294
                     /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
     polyA_signal    181..657
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     439..458
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     misc_feature    689..1277
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             795..806
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     1204..1224
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    1302..1335
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     protein_bind    1386..1419
                     /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    1426..1550
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        1575..1605
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1659..2318
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1725..1744)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2660..2965
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     2844..2863
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(3141..3174)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     protein_bind    complement(3225..3258)
                     /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)."
     promoter        complement(3289..4467)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3298..4236
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(3321..3341)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     repeat_region   4568..4697
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(4759..5347)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4839..4858)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5518..6378)
                     /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     6141..6160
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6379..6483)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6551..6569
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6607..6629)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6729..6748
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(6765..7220)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6902..6923)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7114..7133
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgct cggtccgcac gtggttacct acaaaatcag
      181 aaggacaggg aagggagcag tggttcacgc ctgtaatccc agcaatttgg gaggccaagg
      241 tgggtagatc acctgagatt aggagttgga gaccagcctg gccaatatgg tgaaaccccg
      301 tctctaccaa aaaaacaaaa attagctgag cctggtcatg catgcctgga atcccaacaa
      361 ctcgggaggc tgaggcagga gaatcgcttg aacccaggag gcggagattg cagtgagcca
      421 agattgtgcc actgcactcc agcttggttc ccaatagacc ccgcaggccc tacaggttgt
      481 cttcccaact tgccccttgc tccataccac ccccctccac cccataatat tatagaagga
      541 cacctagtca gacaaaatga tgcaacttaa ttttattagg acaaggctgg tgggcactgg
      601 agtggcaact tccagggcca ggagaggcac tggggagggg tcacagggat gccacccgta
      661 gatctctcga gcagcgctcg gtatcgatgc ggggaggcgg cccaaaggga gatccgactc
      721 gtctgagggc gaaggcgaag acgcggaaga ggccgcagag ccggcagcag gccgcgggaa
      781 ggaaggtccg ctggattgag ggccgaaggg acgtagcaga aggacgtccc gcgcagaatc
      841 caggtggcaa cataggcgag cagccaagga aaggacgatg atttccccga caacaccacg
      901 gaattgtcag tgcccaacag ccgagcccct gtccagcagc gggcaaggca ggcggcgatg
      961 agttccgccg tggcaatagg gagggggaaa gcgaaagtcc cggaaaggag ctgacaggtg
     1021 gtggcaatgc cccaaccagt gggggttgcg tcagcaaaca cagtgcacac cacgccacgt
     1081 tgcctgacaa cgggccacaa ctcctcataa agagacagca accaggattt atacaaggag
     1141 gagaaaatga aagccatacg ggaagcaata gcatgataca aaggcattaa agcagcgtat
     1201 ccacatagcg taaaaggagc aacatagtta agaataccag tcaatctttc acaaattttg
     1261 taatccagag gttgattatc gataagcttg atatcgaatt cataacttcg tatagcatac
     1321 attatacgaa gttattcttt gcaccattct aaagaataac agtgataatt tctgggttaa
     1381 ggcaaataac ttcgtatagg atactttata cgaagttatg ctagcacaag tttgtacaaa
     1441 aaagctgaac gagaaacgta aaatgatata aatatcaata tattaaatta gattttgcat
     1501 aaaaaacaga ctacataata ctgtaaaaca caacatatcc agtcactatg gcggccgcat
     1561 taggcacccc aggctttaca ctttatgctt ccggctcgta taatgtgtgg attttgagtt
     1621 aggatccgtc gagattttca ggagctaagg aagctaaaat ggagaaaaaa atcactggat
     1681 ataccaccgt tgatatatcc caatggcatc gtaaagaaca ttttgaggca tttcagtcag
     1741 ttgctcaatg tacctataac cagaccgttc agctggatat tacggccttt ttaaagaccg
     1801 taaagaaaaa taagcacaag ttttatccgg cctttattca cattcttgcc cgcctgatga
     1861 atgctcatcc ggaattccgt atggcaatga aagacggtga gctggtgata tgggatagtg
     1921 ttcacccttg ttacaccgtt ttccatgagc aaactgaaac gttttcatcg ctctggagtg
     1981 aataccacga cgatttccgg cagtttctac acatatattc gcaagatgtg gcgtgttacg
     2041 gtgaaaacct ggcctatttc cctaaagggt ttattgagaa tatgtttttc gtctcagcca
     2101 atccctgggt gagtttcacc agttttgatt taaacgtggc caatatggac aacttcttcg
     2161 cccccgtttt caccatgggc aaatattata cgcaaggcga caaggtgctg atgccgctgg
     2221 cgattcaggt tcatcatgcc gtttgtgatg gcttccatgt cggcagaatg cttaatgaat
     2281 tacaacagta ctgcgatgag tggcagggcg gggcgtaaac gcgtggatcc ggcttactaa
     2341 aagccagata acagtatgcg tatttgcgcg ctgatttttg cggtataaga atatatactg
     2401 atatgtatac ccgaagtatg tcaaaaagag gtatgctatg aagcagcgta ttacagtgac
     2461 agttgacagc gacagctatc agttgctcaa ggcatatatg atgtcaatat ctccggtctg
     2521 gtaagcacaa ccatgcagaa tgaagcccgt cgtctgcgtg ccgaacgctg gaaagcggaa
     2581 aatcaggaag ggatggctga ggtcgcccgg tttattgaaa tgaacggctc ttttgctgac
     2641 gagaacaggg gctggtgaaa tgcagtttaa ggtttacacc tataaaagag agagccgtta
     2701 tcgtctgttt gtggatgtac agagtgatat tattgacacg cccgggcgac ggatggtgat
     2761 ccccctggcc agtgcacgtc tgctgtcaga taaagtctcc cgtgaacttt acccggtggt
     2821 gcatatcggg gatgaaagct ggcgcatgat gaccaccgat atggccagtg tgccggtctc
     2881 cgttatcggg gaagaagtgg ctgatctcag ccaccgcgaa aatgacatca aaaacgccat
     2941 taacctgatg ttctggggaa tataaatgtc aggctccctt atacacagcc agtctgcagg
     3001 tcgaccatag tgactggata tgttgtgttt tacagtatta tgtagtctgt tttttatgca
     3061 aaatctaatt taatatattg atatttatat cattttacgt ttctcgttca gctttcttgt
     3121 acaaatttag ttggcgcgcc ataacttcgt ataatgtatg ctatacgaag ttattaagag
     3181 gtttcatatt gctaatagca gctacaatcc agctaccatt ctgcataact tcgtataaag
     3241 tatcctatac gaagttattc cggagtcgac tctagaggat ccggtacctc acgacacctg
     3301 aaatggaaga aaaaaacttt gaaccactgt ctgaggcttg agaatgaacc aagatccaaa
     3361 ctcaaaaagg gcaaattcca aggagaatta catcaagtgc caagctggcc taacttcagt
     3421 ctccacccac tcagtgtggg gaaactccat cgcataaaac ccctcccccc aacctaaaga
     3481 cgacgtactc caaaagctcg agaactaatc gaggtgcctg gacggcgccc ggtactccgt
     3541 ggagtcacat gaagcgacgg ctgaggacgg aaaggccctt ttcctttgtg tgggtgactc
     3601 acccgcccgc tctcccgagc gccgcgtcct ccattttgag ctccctgcag cagggccggg
     3661 aagcggccat ctttccgctc acgcaactgg tgccgaccgg gccagccttg ccgcccaggg
     3721 cggggcgata cacggcggcg cgaggccagg caccagagca ggccggccag cttgagacta
     3781 cccccgtccg attctcggtg gccgcgctcg caggccccgc ctcgccgaac atgtgcgctg
     3841 ggacgcacgg gccccgtcgc cgcccgcggc cccaaaaacc gaaataccag tgtgcagatc
     3901 ttggcccgca tttacaagac tatcttgcca gaaaaaaagc gtcgcagcag gtcatcaaaa
     3961 attttaaatg gctagagact tatcgaaagc agcgagacag gcgcgaaggt gccaccagat
     4021 tcgcacgcgg cggccccagc gcccaggcca ggcctcaact caagcacgag gcgaaggggc
     4081 tccttaagcg caaggcctcg aactctccca cccacttcca acccgaagct cgggatcaag
     4141 aatcacgtac tgcagccagg tggaagtaat tcaaggcacg caagggccat aacccgtaaa
     4201 gaggccaggc ccgcgggaac cacacacggc acttacctgt gttctggcgg caaacccgtt
     4261 gcgaaaaaga acgttcacgg cgactactgc acttatatac ggttctcccc caccctcggg
     4321 aaaaaggcgg agccagtaca cgacatcact ttcccagttt accccgcgcc accttctcta
     4381 ggcaccggtt caattgccga cccctccccc caacttctcg gggactgtgg gcgatgtgcg
     4441 ctctgcccac tgacgggcac cggagccaat tcccactcct ttcaagacct agaaggtcca
     4501 ttagctgcaa agattcctct ctgtttaaaa ctttatccat ctttgcaaag cttacgcgtg
     4561 cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc
     4621 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggcg gcctcagtga gcgagcgagc
     4681 gcgcagctgc ctgcaggaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     4741 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     4801 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     4861 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     4921 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     4981 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     5041 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     5101 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     5161 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     5221 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5281 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5341 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     5401 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     5461 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     5521 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     5581 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     5641 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     5701 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     5761 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     5821 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     5881 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     5941 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     6001 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     6061 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     6121 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     6181 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     6241 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     6301 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     6361 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     6421 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     6481 gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt
     6541 aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg
     6601 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
     6661 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct
     6721 taactatgcg gcatcagagc agattgtact gagagtgcac cataaaattg taaacgttaa
     6781 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaatagac
     6841 cgaaatcggc aaaatccctt ataaatcaaa agaatagccc gagatagagt tgagtgttgt
     6901 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     6961 aaccgtctat cagggcgatg gcccactacg tgaaccatca cccaaatcaa gttttttggg
     7021 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     7081 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     7141 taaggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     7201 tgcgccgcta cagggcgcgt actatggttg ctttgacgta tgcggtgtga aataccgcac
     7261 agatgcgtaa ggagaaaata ccgcatcagg cgcc
//
