LOCUS       Exported                6359 bp ds-DNA     circular SYN 28-APR-2022
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    C.55-pAAV-Syn-GFP-IRES-hSNCA (A11P/V70P)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6359)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Apr 28, 2022 from SnapGene 4.3.11
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6359
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          664..665
                     /organism="synthetic DNA construct"
                     /plasmid="NEBuilder_Assembly"
                     /note="color: #ffffff"
     source          664..665
                     /organism="synthetic DNA construct
                     /mol_type=""other DNA
                     /note=""pCAG"
                     /note="color: #ffffff"
     source          672..2032
                     /organism="synthetic DNA construct"
                     /plasmid="NEBuilder_Assembly"
                     /note="color: #ffffff"
     source          672..1401
                     /organism="synthetic DNA construct
                     /mol_type=""other DNA
                     /note=""pCAG"
                     /note="color: #ffffff"
     source          1402..2026
                     /organism="synthetic DNA construct
                     /mol_type=""other DNA
                     /note=""IRES"
                     /note="color: #ffffff"
     source          2027..2032
                     /organism="synthetic DNA construct
                     /mol_type=""other DNA
                     /note=""mSnca"
                     /note="color: #ffffff"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
                     /note="color: #ffe4c4"
     primer_bind     complement(23..34)
                     /label=L4440
                     /note="L4440 vector, forward primer"
                     /note="color: black; sequence: AGCGAGTCAGTGAGCGAG"
     primer_bind     84..95
                     /label=L4440
                     /note="L4440 vector, forward primer"
                     /note="color: black; sequence: AGCGAGTCAGTGAGCGAG"
     primer_bind     150..173
                     /label=MluI-Syn-F
                     /note="color: black; sequence: ATTACGCGTtaactgcagagggccctg"
     promoter        180..627
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
                     /note="color: #ffffff; direction: RIGHT"
     primer_bind     509..527
                     /label=Syn-Seqa
                     /note="color: black; sequence: ACGCGAGGCGCGAGATAGG"
     primer_bind     509..527
                     /label=Syn-Seq
                     /note="color: black; sequence: acgcgaggcgcgagatagg"
     regulatory      673..682
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /note="color: #a6acb3"
     CDS             679..1398
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /note="This feature has 3 segments:
                        1:  679 ..  681 / #05fd14
                        2:  682 ..  684 / #05fd14 / 1a
                        3:  685 .. 1398 / #05fd14"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(724..745)
                     /label=EGFP-N
                     /note="color: black; sequence: CGTCGCCGTCCAGCTCGACCAG"
     primer_bind     1332..1353
                     /label=EGFP-C
                     /note="color: black; sequence: CATGGTCCTGCTGGAGTTCGTG"
     primer_bind     complement(1376..1404)
                     /label=GFP-rev
                     /note="color: black; sequence: 
                     ctggctttacttgtacagctcgtccatgc"
     primer_bind     1385..1433
                     /label=IRES_fwd
                     /note="3'Tm=76.1 3'Ta(annealing temp)=72.0"
                     /note="color: black; sequence: 
                     agctgtacaagtaaagcCAGTGTGGTGGTACGTAGGAATTCTCCCTCCC"
     misc_feature    1426..1992
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
                     /note="color: #a6acb3"
     primer_bind     complement(1515..1534)
                     /label=IRES-Ra
                     /note="color: black; sequence: CCTCACATTGCCAAAAGACG"
     primer_bind     complement(1515..1534)
                     /label=IRES-R
                     /note="color: black; sequence: cctcacattgccaaaagacg"
     primer_bind     1814..1833
                     /label=IRES-F
                     /note="color: black; sequence: TGGCTCTCCTCAAGCGTATT"
     primer_bind     2021..2054
                     /label=SalI-hSNCA-F
                     /note="color: black; sequence: 
                     ATgtcgacgccaccATGGATGTATTCATGAAAGGAC"
     regulatory      2027..2036
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /note="color: #a6acb3"
     regulatory      2027..2032
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /note="color: #a6acb3"
     CDS             2033..2455
                     /codon_start=1
                     /label=hSNCA
                     /note="color: #00ccff"
                     /translation="MDVFMKGLSKPKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTK
                     EGVVHGVATVAEKTKEQVTNVGGAPVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNE
                     EGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA"
     variation       2064..2065
                     /label=A11P (GCC>CCC)
                     /note="color: #ff0000"
     variation       2242
                     /label=V70P (GTG>CCG)
                     /note="color: #ff0000"
     primer_bind     complement(2437..2461)
                     /label=NheI-hSNCA
                     /note="color: black; sequence: 
                     ATAgctagcTTAGGCTTCAGGTTCGTAG"
     misc_feature    2486..3074
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
                     /note="color: #ffffff"
     primer_bind     complement(2539..2559)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
                     /note="color: black; sequence: CATAGCGTAAAAGGAGCAACA"
     primer_bind     complement(2699..2718)
                     /label=seq-wpre-R (Sui)
                     /note="sw primer box2 #1392"
                     /note="color: black; sequence: gttgcgtcagcaaacacagt"
     CDS             complement(2957..2968)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /note="color: #cc99b2
                     Cleavage site after base 2956"
                     /translation="IEGR"
     misc_feature    3088..3090
                     /label=AfeI
                     /note="color: #ff99cc"
     polyA_signal    3106..3582
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
                     /note="color: #a6acb3"
     primer_bind     complement(3119..3136)
                     /label=bGH-Ra
                     /note="color: black; sequence: GCACTGGGGAGGGGTCAC"
     primer_bind     complement(3119..3136)
                     /label=bGH-R
                     /note="color: black; sequence: gcactggggaggggtcac"
     repeat_region   3622..3762
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
                     /note="color: #ffe4c4"
     primer_bind     complement(3668..3679)
                     /label=L4440
                     /note="L4440 vector, forward primer"
                     /note="color: black; sequence: AGCGAGTCAGTGAGCGAG"
     primer_bind     3729..3740
                     /label=L4440
                     /note="L4440 vector, forward primer"
                     /note="color: black; sequence: AGCGAGTCAGTGAGCGAG"
     rep_origin      3837..4292
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     primer_bind     complement(3924..3943)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
                     /note="color: black; sequence: AGGGAAGAAAGCGAAAGGAG"
     primer_bind     4134..4155
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
                     /note="color: black; sequence: GTGGACTCTTGTTCCAAACTGG"
     promoter        4574..4678
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: RIGHT"
     CDS             4679..5539
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                        1: 4679 .. 4747 / #ccffcc / signal sequence
                        2: 4748 .. 5539 / #ccffcc
                     Cleavage site after base 4747"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4897..4916)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
                     /note="color: black; sequence: ATAATACCGCGCCACATAGC"
     rep_origin      5710..6298
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     primer_bind     6199..6218
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
                     /note="color: black; sequence: GGGAAACGCCTGGTATCTTT"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgttaact gcagagggcc ctgcgtatga
      181 gtgcaagtgg gttttaggac caggatgagg cggggtgggg gtgcctacct gacgaccgac
      241 cccgacccac tggacaagca cccaaccccc attccccaaa ttgcgcatcc cctatcagag
      301 agggggaggg gaaacaggat gcggcgaggc gcgtgcgcac tgccagcttc agcaccgcgg
      361 acagtgcctt cgcccccgcc tggcggcgcg cgccaccgcc gcctcagcac tgaaggcgcg
      421 ctgacgtcac tcgccggtcc cccgcaaact ccccttcccg gccaccttgg tcgcgtccgc
      481 gccgccgccg gcccagccgg accgcaccac gcgaggcgcg agataggggg gcacgggcgc
      541 gaccatctgc gctgcggcgc cggcgactca gcgctgcctc agtctgcggt gggcagcgga
      601 ggagtcgtgt cgtgcctgag agcgcgtccg gagctattag caatatgaaa cctcttaggc
      661 gcgccaccgg tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
      721 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
      781 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
      841 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
      901 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
      961 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     1021 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     1081 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     1141 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     1201 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     1261 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
     1321 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     1381 gacgagctgt acaagtaaag ccagtgtggt ggtacgtagg aattctccct cccccccccc
     1441 taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct atatgttatt
     1501 ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt
     1561 gacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc tgttgaatgt
     1621 cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa acaacgtctg tagcgaccct
     1681 ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa agccacgtgt
     1741 ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt ggatagttgt
     1801 ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg atgcccagaa
     1861 ggtaccccat tgtatgggat ctgatctggg gcctcggtgc acatgcttta cgtgtgttta
     1921 gtcgaggtta aaaaacgtct aggccccccg aaccacgggg acgtggtttt cctttgaaaa
     1981 acacgatgat aatggtaccg cgggcccggg atcctctaga gtcgacgcca ccatggatgt
     2041 attcatgaaa ggactttcaa agcccaagga gggagttgtg gctgctgctg agaaaaccaa
     2101 acagggtgtg gcagaagcag caggaaagac aaaagagggt gttctctatg taggctccaa
     2161 aaccaaggag ggagtggtgc atggtgtggc aacagtggct gagaagacca aagagcaagt
     2221 gacaaatgtt ggaggagcac cggtgacggg tgtgacagca gtagcccaga agacagtgga
     2281 gggagcaggg agcattgcag cagccactgg ctttgtcaaa aaggaccagt tgggcaagaa
     2341 tgaagaagga gccccacagg aaggaattct ggaagatatg cctgtggatc ctgacaatga
     2401 ggcttatgaa atgccttctg aggaagggta tcaagactac gaacctgaag cctaagctag
     2461 cgaattcgat atcaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt
     2521 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     2581 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     2641 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac
     2701 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc
     2761 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc
     2821 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa
     2881 atcatcgtcc tttccttggc tgctcgccta tgttgccacc tggattctgc gcgggacgtc
     2941 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc
     3001 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg
     3061 ggccgcctcc ccgcatcgat accgagcgct gctcgagaga tctacgggtg gcatccctgt
     3121 gacccctccc cagtgcctct cctggccctg gaagttgcca ctccagtgcc caccagcctt
     3181 gtcctaataa aattaagttg catcattttg tctgactagg tgtccttcta taatattatg
     3241 gggtggaggg gggtggtatg gagcaagggg caagttggga agacaacctg tagggcctgc
     3301 ggggtctatt gggaaccaag ctggagtgca gtggcacaat cttggctcac tgcaatctcc
     3361 gcctcctggg ttcaagcgat tctcctgcct cagcctcccg agttgttggg attccaggca
     3421 tgcatgacca ggctcagcta atttttgttt ttttggtaga gacggggttt caccatattg
     3481 gccaggctgg tctccaactc ctaatctcag gtgatctacc caccttggcc tcccaaattg
     3541 ctgggattac aggcgtgaac cactgctccc ttccctgtcc ttctgatttt gtaggtaacc
     3601 acgtgcggac cgagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc
     3661 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc
     3721 gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt
     3781 ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc
     3841 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     3901 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     3961 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     4021 tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc
     4081 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     4141 cttgttccaa actggaacaa cactcaaccc tatctcgggc tattcttttg atttataagg
     4201 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     4261 gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc
     4321 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     4381 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     4441 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     4501 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     4561 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     4621 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     4681 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     4741 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     4801 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     4861 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     4921 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     4981 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     5041 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     5101 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     5161 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     5221 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     5281 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     5341 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     5401 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     5461 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     5521 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     5581 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     5641 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     5701 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     5761 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     5821 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     5881 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     5941 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     6001 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     6061 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     6121 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     6181 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     6241 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     6301 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt
//
