LOCUS       Exported                7057 bp DNA     circular SYN 27-SEP-2019
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 7057)
  AUTHORS   Fishell Lab
  TITLE     Direct Submission
  JOURNAL   Exported Nov 3, 2021 from SnapGene 5.3.2
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7057
                     /organism="recombinant plasmid"
                     /mol_type="other DNA"
     misc_feature    1..130
                     /label=ITR
     misc_feature    131..608
                     /label=hSyn1 Promoter
     misc_feature    138..149
                     /label=To synth
     misc_feature    144..149
                     /label=ApaI
     misc_feature    150..761
                     /label=To synth
     primer_bind     519..536
                     /label=Seq-hSyn
     misc_feature    609..628
                     /label=P/E cut
     misc_feature    609..622
                     /label=140708_Sequencing_Clone1
                     /note="Check 140708_AAV_GFP_1_G_EGFP-N"
     misc_feature    609..622
                     /label=140708_Sequencing_Clone1
                     /note="Check 140708_AAV_GFP_1_G_EGFP-N"
     misc_feature    623..761
                     /label=140708_Sequencing_Clone1
                     /note="Check 140708_AAV_GFP_1_R_140619_Seq_AAV_Dlx_R"
     misc_feature    623..760
                     /label=From_JD6
     misc_feature    623..636
                     /label=Gibs_Flex_Fb
     protein_bind    629..662
                     /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."
     protein_bind    713..746
                     /label=LoxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    735..754
                     /label=Gibson_Flex_R1
     primer_bind     746..792
                     /label=GA-BFP2.R
     misc_feature    755..760
                     /label=140708_Sequencing_Clone1
                     /note="Check 140708_AAV_GFP_1_G_EGFP-N"
     misc_feature    755..760
                     /label=SpeI
     misc_feature    761
                     /label=MCS
                     /note=" multiple cloning site
                     "
     misc_feature    767..1465
                     /label=PCR_Insert2
     CDS             complement(767..1465)
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     primer_bind     767..791
                     /label=Gibs_Puncta_R2
     misc_feature    767..788
                     /label=Ra
     primer_bind     768..794
                     /label=GA-BFP.Ra
     primer_bind     complement(790..811)
                     /label=Rec-BFP.F
     primer_bind     complement(1442..1473)
                     /label=GA-BFP2.F
     primer_bind     complement(1442..1465)
                     /label=Gibs_Puncta_F2
     misc_feature    1446..1465
                     /label=Rb
     primer_bind     complement(1447..1465)
                     /label=GA-BFP.Fa
     primer_bind     1459..1484
                     /label=GA-GpGi.R
     misc_feature    1466..3346
                     /label=140708_Sequencing_Clone1
                     /note="Check 140708_AAV_GFP_1_R_140619_Seq_AAV_Dlx_R"
     CDS             complement(1466..1531)
                     /codon_start=1
                     /label=P2A
                     /translation="GSGATNFSLLKQAGDVEENPGP"
     primer_bind     complement(1518..1563)
                     /label=Gibson_NLS_F2
     primer_bind     1518..1563
                     /label=Gibson_NLS_R1
     misc_feature    1518..1537
                     /label=Gibson_NLS_F2
     misc_feature    1538..1543
                     /label=BspEI
     CDS             complement(1544..3340)
                     /codon_start=1
                     /product="From Roth LabHA tag added
                     "
                     /label=hM3Dq
                     /translation="MYPYDVPDYATLHNNSTTSPLFPNISSSWIHSPSDAGLPPGTVTH
                     FGSYNVSRAAGNFSSPDGTTDDPLGGHTVWQVVFIAFLTGILALVTIIGNILVIVSFKV
                     NKQLKTVNNYFLLSLACADLIIGVISMNLFTTYIIMNRWALGNLACDLWLAIDCVASNA
                     SVMNLLVISFDRYFSITRPLTYRAKRTTKRAGVMIGLAWVISFVLWAPAILFWQYFVGK
                     RTVPPGECFIQFLSEPTITFGTAIAGFYMPVTIMTILYWRIYKETEKRTKELAGLQASG
                     TEAETENFVHPTGSSRSCSSYELQQQSMKRSNRRKYGRCHFWFTTKSWKPSSEQMDQDH
                     SSSDSWNNNDAAASLENSASSDEEDIGSETRAIYSIVLKLPGHSTILNSTKLPSSDNLQ
                     VPEEELGMVDLERKADKLQAQKSVDDGGSFPKSFSKLPIQLESAVDTAKTSDVNSSVGK
                     STATLPLSFKEATLAKRFALKTRSQITKRKRMSLVKEKKAAQTLSAILLAFIITWTPYN
                     IMVLVNTFCDSCIPKTFWNLGYWLCYINSTVNPVCYALCNKTFRTTFKMLLLCQCDKKK
                     RRKQQYQQRQSVIFHKRAPEQAL"
     misc_feature    1544..1563
                     /label=Gibson_NLS_R1
     misc_feature    1544..1556
                     /label=Overlap Gibson 2A
     misc_feature    1544..1546
                     /label=150212_AAV_hDLX_F7_Sequenced
                     /note="Ok for the 12 minis"
     primer_bind     complement(1583..1602)
                     /label=Seq_AAV_hDlx_F7
     primer_bind     complement(1791..1810)
                     /label=Seq_AAV_hDlx_F6
     misc_feature    1949
                     /label=End of sequencing Clone 1 R
     misc_feature    2144
                     /label=End of sequencing Clone1
     misc_feature    2225
                     /label=Missing in Clone 3 F
                     /note="Could be double peak on Raw"
     primer_bind     2256..2273
                     /label=Seq-Gq-midR
     misc_feature    2273
                     
     primer_bind     complement(2383..2402)
                     /label=Seq_AAV_hDlx_F5
     primer_bind     complement(2537..2562)
                     /label=Seq-Gq.m.F
     primer_bind     complement(2912..2931)
                     /label=Seq_AAV_hDlx_F4
     primer_bind     complement(3291..3371)
                     /label=Gibson_HA-NLS_F1
     misc_feature    3291..3310
                     /label=Gibson_NLS_F1b
     misc_feature    3311..3337
                     /label=140708_Sequencing_Clone1
                     /note="Check 140708_AAV_GFP_1_R_140619_Seq_AAV_Dlx_R"
     misc_feature    3311..3337
                     /label=HA-Tag
     primer_bind     complement(3323..3367)
                     /label=GA-Gp.F
     misc_feature    3341..3346
                     /label=Kozak
     misc_feature    3352..3489
                     /label=140708_Sequencing_Clone1
                     /note="Check 140708_AAV_GFP_1_R_140619_Seq_AAV_Dlx_R"
     misc_feature    3352..3489
                     /label=To synth
     misc_feature    3352
                     /label=MCS
                     /note=" multiple cloning site
                     "
     misc_feature    3353..3489
                     /label=From_JD6
     misc_feature    3353..3359
                     /label=140708_Sequencing_Clone1
                     /note="Check 140708_AAV_GFP_1_G_EGFP-N"
     misc_feature    3354..3359
                     /label=SpeI
     misc_feature    3372..3405
                     /label=140708_Sequencing_Clone1
                     /note="Check 140708_AAV_GFP_1_R_140619_Seq_AAV_Dlx_R"
     protein_bind    complement(3372..3405)
                     /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."
     misc_feature    3398..3405
                     /label=Gibs_Flex_Fb
     misc_feature    3456..3489
                     /label=140708_Sequencing_Clone1
                     /note="Check 140708_AAV_GFP_1_R_140619_Seq_AAV_Dlx_R"
     protein_bind    complement(3456..3489)
                     /label=LoxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    3456..3467
                     /label=Gibson_Flex_R1
     primer_bind     complement(3488..3502)
                     /label=Gibs_Puncta_R2
     primer_bind     complement(3490..3515)
                     /label=GA-BFP.Ra
     misc_feature    3490..3503
                     /label=To synth
     misc_feature    3490..3497
                     /label=AscI
     misc_feature    3498..4086
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3564..3583)
                     /label=Seq-WPRE5-R
     polyA_signal    4094..4318
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4320..4460
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      4535..4990
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        5272..5376
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5377..6237
                     /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"
     rep_origin      6408..6996
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gctagactgc agagggccct gcgtatgagt gcaagtgggt tttaggacca
      181 ggatgaggcg gggtgggggt gcctacctga cgaccgaccc cgacccactg gacaagcacc
      241 caacccccat tccccaaatt gcgcatcccc tatcagagag ggggagggga aacaggatgc
      301 ggcgaggcgc gtgcgcactg ccagcttcag caccgcggac agtgccttcg cccccgcctg
      361 gcggcgcgcg ccaccgccgc ctcagcactg aaggcgcgct gacgtcactc gccggtcccc
      421 cgcaaactcc ccttcccggc caccttggtc gcgtccgcgc cgccgccggc ccagccggac
      481 cgcaccacgc gaggcgcgag ataggggggc acgggcgcga ccatctgcgc tgcggcgccg
      541 gcgactcagc gctgcctcag tctgcggtgg gcagcggagg agtcgtgtcg tgcctgagag
      601 cgcagtcaga tatcgtttaa actccggaat aacttcgtat aggatacttt atacgaagtt
      661 atgcagaatg gtagctggat tgtagctgct attagcaata tgaaacctct taataacttc
      721 gtatagcata cattatacga agttataacg cgccactagt gaattcttaa ttaagcttgt
      781 gccccagttt gctagggagg tcgcagtatc tggccactgc cacctcgtgc tgctcgacgt
      841 aggtctcgtt gttggcctcc ttgattcttt ccagtctgta gtccacatag tagacgccag
      901 gcatcttgag gttcttagcg ggtttcttgg atctatatgt ggtcttgatg tttgcgatca
      961 gatggctccc gcccacgagc ttcagggcca tgtcgtttct gccttccagg ccgccgtcag
     1021 cggggtacag cgtctcggtg aaggcctccc agccgagtgt tttcttctgc atcacagggc
     1081 cgttggatgt gaagttcacc cctctgatct tgacgttgta gatgaggcag ccgtcctgga
     1141 ggctggtgtc ctgggtagcg gtcagcacgc ccccgtcttc gtatgtggtg actctctccc
     1201 atgtgaagcc ctcagggaag gactgcttga agaagtcggg gatgccctgg gtgtggttga
     1261 tgaaggtctt gctgccgtag aggaagctag tagccaggat gtcgaaggcg aaggggagag
     1321 ggccgccctc gaccaccttg attctcatgg tctgggtgcc ctcgtagggc ttgccttcgc
     1381 cctcggatgt gcacttgaag tgatggttgt ccacggtgcc ctccatgtac agcttcatgt
     1441 gcatgttctc cttaatcagc tcgcttggcc cgggattctc ttcgacatcc cctgcttgtt
     1501 tcaacaggga gaagttagtg gctccgcttc cgttatctcc ggacaaggcc tgctcgggtg
     1561 cgcgcttgtg aaaaatgacc gactgtctct gctggtactg ctgcttgcgc ctcttttttt
     1621 tgtcacactg gcacagcagc agcatcttga aagtggttct gaatgttttg ttgcacagag
     1681 catagcacac ggggttcacg gtgctgttga tgtagcacag ccagtagccc agattccaaa
     1741 aggttttggg tatgcagctg tcacaaaagg tgttcaccag aaccatgatg ttgtatgggg
     1801 tccaagtgat gatgaaggca agcaagatcg cactgagggt ctgggccgct ttcttctcct
     1861 tgaccaggga catccttttc cgcttagtga tctgacttct ggtcttcaga gcaaacctct
     1921 tggccagagt ggcttccttg aaggacagag gtagagtggc cgtgctctta cccactgagg
     1981 agttgacgtc agaagtctta gctgtgtcca cggctgactc tagctggatg ggaagcttgg
     2041 agaagctttt tggaaaactg cctccatcgt ccacgctctt ctgggcctgc agcttgtcgg
     2101 ctttcctctc caagtccacc atccccagct cctcctcagg cacctgcagg ttgtccgatg
     2161 agggtaactt ggtggagttg aggatggtgc tgtgacccgg aagcttgagc acgatggagt
     2221 agatggctct cgtctcggag ccaatgtcct cctcgtcgga ggaggcggag ttctccaggg
     2281 aggcagcagc atcattgttg ttccaactgt cactgctgct gtggtcttgg tccatctgct
     2341 cggagctggg tttccagctc ttggttgtga accagaagtg gcagcggcca tacttcctcc
     2401 tgttggagcg tttcatgctt tgctgttgaa gttcgtaact gctgcagctt cgagaactgc
     2461 ccgtggggtg gacaaagttt tctgtctctg cctctgtccc agaggcttgc aggccagcaa
     2521 gctctttggt acgcttttca gtttccttat agatcctcca gtataaaata gtcataatgg
     2581 tgacaggcat ataaaaacca gcgatggctg tgccaaaagt aatggtgggc tcactgagga
     2641 actgaatgaa gcactctccc ggaggcacag ttctctttcc aacaaagtat tgccagaaca
     2701 agatggcagg agcccaaagg acaaaggaga tgacccaagc cagaccgatc atcacaccgg
     2761 ctctctttgt tgttcgtttg gctcggtacg tgagcggcct cgtgatggaa aagtatctgt
     2821 caaagctgat gaccagaaga ttcataacag aggcattgct ggctacgcag tcaatggcaa
     2881 gccagaggtc acaggccaag ttccctaagg cccatcgatt catgatgatg taggtcgtaa
     2941 acagattcat tgaaatgacc ccgataatca gatcggcaca ggccaggctt aagaggaagt
     3001 agttgttgac cgtcttcagc tgcttgttga ccttaaatga cacaattacc aggatgttgc
     3061 cgatgatggt caccaaggcc aggatgcccg ttaagaaagc gatgaagacc acttgccaga
     3121 cggtatgacc tcccagaggg tcatcggtgg taccgtctgg agaggagaaa ttgccagctg
     3181 ctcgagaaac attgtagctg ccgaaatgag tgacggttcc cgggggcagc cctgcatcgg
     3241 aggggctgtg tatccaggag gagctgatgt ttggaaacaa aggcgaggtt gtactgttat
     3301 tgtgcaaggt agcgtaatct ggaacatcgt atgggtacat ggtggcaagc tttactagtg
     3361 gtggcgctag cataacttcg tataaagtat cctatacgaa gttatttgcc ttaacccaga
     3421 aattatcact gttattcttt agaatggtgc aaagaataac ttcgtataat gtatgctata
     3481 cgaagttatg gcgcgccaat caacctctgg attacaaaat ttgtgaaaga ttgactggta
     3541 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc
     3601 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt
     3661 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg
     3721 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt
     3781 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct
     3841 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aagctgacgt
     3901 cctttccatg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct
     3961 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc
     4021 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct
     4081 ccccgcctgg atcctgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     4141 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     4201 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     4261 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggca
     4321 ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc
     4381 cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg
     4441 agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     4501 tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc
     4561 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     4621 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     4681 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     4741 aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     4801 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     4861 ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     4921 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     4981 tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     5041 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     5101 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     5161 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     5221 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     5281 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     5341 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     5401 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     5461 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     5521 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     5581 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     5641 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     5701 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     5761 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     5821 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     5881 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     5941 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     6001 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     6061 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     6121 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     6181 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     6241 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     6301 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     6361 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     6421 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     6481 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     6541 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     6601 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     6661 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     6721 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     6781 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     6841 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     6901 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     6961 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     7021 cggttcctgg ccttttgctg gccttttgct cacatgt
//
