LOCUS       Exported                8555 bp ds-DNA     circular SYN 21-DEC-2020
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8555)
  AUTHORS   mao-s
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, Dec 22, 2020 from SnapGene Viewer 5.2.4
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8555
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      complement(57..642)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(813..1673)
                     /label=Amp(R)
     promoter        complement(1674..1772)
                     /label=bla promoter
     promoter        2041..2628
                     /label=CMV promoter
     misc_feature    2567..2585
                     /label=CGCCCCATTGACGCAAATG
     misc_feature    2567..2585
                     /label=CMV For
     misc_feature    2578..2598
                     /label=CMV forward primer
     misc_feature    2631..2655
                     /label=TGGCTAACTAGAGAACCCACTGCTT
     misc_feature    2672..2691
                     /label=T7 primer
     promoter        2672..2688
                     /label=T7 promoter
     CDS             2735..2788
                     /codon_start=1
                     /label=pbnls
                     /translation="KRTADGSEFESPKKKRKV"
     misc_feature    2795..3346
                     /label=frag1
     CDS             2795..3346
                     /codon_start=1
                     /label=MOUSE dCas13d
                     /translation="IEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDSI
                     RSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEK
                     RYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIGFGKFSTVY
                     TYDEFKDPEHHRAAFNNNDKL"
     variation       3326..3346
                     /label=delete in 1023b
     CDS             3353..3406
                     /codon_start=1
                     /label=pbnls
                     /translation="KRTADGSEFESPKKKRKV"
     misc_feature    3413..4567
                     /label=huADAR2
     misc_feature    3753..3758
                     /label=566-567
     misc_feature    3929..3931
                     /label=E to Q muatnt
     CDS             4628..6973
                     /codon_start=1
                     /label=MOUSE dCas13d
                     /translation="INAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYIINYGNECYD
                     ILALLSGLAHWVVANNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNELTNSFSK
                     NSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRKDMSEIRKN
                     HKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFVINLRGSFN
                     DDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAGRDVSAFS
                     KLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFFKDSAKIA
                     DELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDENGNKLKKG
                     KHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQKKQGQNGK
                     NQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGRENAEREKFK
                     KIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEEKGFSSVT
                     KLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKKAEEFTRQI
                     NREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFANKAVALEVARYVHAYINDIAE
                     VNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCVPFGYCIPR
                     FKNLSIEALFDRNEAAKFDKEKKKVSGNS"
     misc_feature    4628..5040
                     /label=frag1
     variation       4628..4651
                     /label=delete in 1023b
     misc_feature    5041..6017
                     /label=frag2
     variation       5207..5248
                     /label=delete in 1023c
     misc_feature    5750..5833
                     /label=delete in 1070/1023a
     CDS             6980..7033
                     /codon_start=1
                     /label=pbnls
                     /translation="KRTADGSEFESPKKKRKV"
     CDS             7040..7105
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             7130..7186
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             7193..7903
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     CDS             7193..7900
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     misc_feature    7213..7241
                     /label=Feature 30
     misc_feature    7412..7444
                     /label=Feature 30
     misc_feature    7632..7662
                     /label=Feature 31
                     /note="secondary structure"
     misc_feature    complement(7904..7933)
                     /label=NotI GH
     misc_feature    complement(7904..7905)
                     /label=NotI
     misc_feature    7912..7915
                     /label=Golden Gate
     misc_feature    complement(7942..7959)
                     /label=BGH reverse primer
     polyA_site      7948..8172
                     /label=BGH pA
     polyA_signal    7948..8172
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    complement(7984..8002)
                     /label=AAGGAAGGCACGGGGGAGG
     primer_bind     complement(8210..8226)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    8234..8250
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(8258..8288)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8303..8324
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(join(8553..8555,1..668))
                     /direction=LEFT
                     /label=pUC origin
ORIGIN
        1 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
       61 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
      121 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
      181 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
      241 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
      301 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
      361 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
      421 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
      481 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
      541 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggttttttt
      601 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
      661 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
      721 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
      781 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
      841 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
      901 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
      961 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     1021 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     1081 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     1141 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     1201 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     1261 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     1321 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     1381 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     1441 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     1501 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     1561 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     1621 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     1681 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     1741 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     1801 cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct
     1861 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg
     1921 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga
     1981 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc
     2041 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
     2101 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     2161 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     2221 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     2281 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     2341 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     2401 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
     2461 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
     2521 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
     2581 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta
     2641 gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag
     2701 ctggctagcg tttaaactta agcttgccac catgaaacgg actgctgacg gcagcgaatt
     2761 tgagagcccc aagaagaaga ggaaagtcgg atccatcgag aagaagaaga gcttcgccaa
     2821 gggcatggga gtgaagagca ccctggtgtc cggctctaag gtgtacatga ccacatttgc
     2881 tgagggaagc gacgccaggc tggagaagat cgtggagggc gatagcatca gatccgtgaa
     2941 cgagggagag gctttcagcg ccgagatggc tgacaagaac gctggctaca agatcggaaa
     3001 cgccaagttt tcccacccaa agggctacgc cgtggtggct aacaacccac tgtacaccgg
     3061 accagtgcag caggacatgc tgggactgaa ggagacactg gagaagaggt acttcggcga
     3121 gtccgccgac ggaaacgata acatctgcat ccaggtcatc cacaacatcc tggatatcga
     3181 gaagatcctg gctgagtaca tcacaaacgc cgcttacgcc gtgaacaaca tctccggcct
     3241 ggacaaggat atcatcggct tcggaaagtt ttctaccgtg tacacatacg acgagttcaa
     3301 ggatccagag caccaccggg ccgcttttaa caacaacgac aagctgggtt ctaagcgcac
     3361 cgccgatgga tccgagttcg aatcccctaa aaagaaaaga aaggtgggat cccagctgca
     3421 tttaccgcag gttttagctg acgctgtctc acgcctggtc ctgggtaagt ttggtgacct
     3481 gaccgacaac ttctcctccc ctcacgctcg cagaaaagtg ctggctggag tcgtcatgac
     3541 aacaggcaca gatgttaaag atgccaaggt gataagtgtt tctacaggaa caaaatgtat
     3601 taatggtgaa tacatgagtg atcgtggcct tgcattaaat gactgccatg cagaaataat
     3661 atctcggaga tccttgctca gatttcttta tacacaactt gagctttact taaataacaa
     3721 agatgatcaa aaaagatcca tctttcagaa atcagagcga ggggggttta ggctgaagga
     3781 gaatgtccag tttcatctgt acatcagcac ctctccctgt ggagatgcca gaatcttctc
     3841 accacatgag ccaatcctgg aagaaccagc agatagacac ccaaatcgta aagcaagagg
     3901 acagctacgg accaaaatag agtctggtca ggggacgatt ccagtgcgct ccaatgcgag
     3961 catccaaacg tgggacgggg tgctgcaagg ggagcggctg ctcaccatgt cctgcagtga
     4021 caagattgca cgctggaacg tggtgggcat ccagggatca ctgctcagca ttttcgtgga
     4081 gcccatttac ttctcgagca tcatcctggg cagcctttac cacggggacc acctttccag
     4141 ggccatgtac cagcggatct ccaacataga ggacctgcca cctctctaca ccctcaacaa
     4201 gcctttgctc agtggcatca gcaatgcaga agcacggcag ccagggaagg cccccaactt
     4261 cagtgtcaac tggacggtag gcgactccgc tattgaggtc atcaacgcca cgactgggaa
     4321 ggatgagctg ggccgcgcgt cccgcctgtg taagcacgcg ttgtactgtc gctggatgcg
     4381 tgtgcacggc aaggttccct cccacttact acgctccaag attaccaagc ccaacgtgta
     4441 ccatgagtcc aagctggcgg caaaggagta ccaggccgcc aaggcgcgtc tgttcacagc
     4501 cttcatcaag gcggggctgg gggcctgggt ggagaagccc accgagcagg accagttctc
     4561 actcacgaaa agaactgcgg atggatctga gtttgagagt ccaaaaaaga agaggaaggt
     4621 cggttctatc aacgccatca aggctcagta cgacgagttc gataactttc tggataaccc
     4681 caggctgggc tacttcggac aggctttctt ttctaaggag ggcagaaact acatcatcaa
     4741 ctacggaaac gagtgttacg acatcctggc cctgctgagc ggactggccc actgggtggt
     4801 ggccaacaac gaggaggagt ctcggatcag ccgcacctgg ctgtacaacc tggacaagaa
     4861 cctggataac gagtacatct ccacactgaa ctacctgtac gacaggatca ccaacgagct
     4921 gacaaacagc ttctccaaga actctgccgc taacgtgaac tacatcgctg agaccctggg
     4981 catcaaccca gctgagttcg ctgagcagta cttcagattt tccatcatga aggagcagaa
     5041 gaacctgggc ttcaacatca caaagctgag agaagtgatg ctggacagaa aggatatgtc
     5101 cgagatcagg aagaaccaca aggtgttcga ttctatcaga accaaggtgt acacaatgat
     5161 ggactttgtg atctacaggt actacatcga ggaggatgcc aaggtggccg ctgccaacaa
     5221 gagcctgccc gacaacgaga agtctctgag cgagaaggat atcttcgtga tcaacctgag
     5281 aggctccttt aacgacgatc agaaggacgc tctgtactac gatgaggcca acaggatctg
     5341 gagaaagctg gagaacatca tgcacaacat caaggagttc cggggaaaca agacccgcga
     5401 gtacaagaag aaggacgctc caaggctgcc taggatcctg cctgctggaa gggacgtgag
     5461 cgccttcagc aagctgatgt acgccctgac aatgtttctg gacggaaagg agatcaacga
     5521 tctgctgacc acactgatca acaagttcga caacatccag tcttttctga aagtgatgcc
     5581 tctgatcggc gtgaacgcta agttcgtgga ggagtacgcc ttctttaagg acagcgccaa
     5641 gatcgctgat gagctgcggc tgatcaagtc ctttgccagg atgggagagc caatcgctga
     5701 cgctaggaga gctatgtaca tcgatgccat ccggatcctg ggaaccaacc tgtcttacga
     5761 cgagctgaag gctctggccg acaccttcag cctggatgag aacggcaaca agctgaagaa
     5821 gggcaagcac ggaatgcgca acttcatcat caacaacgtg atcagcaaca agcggtttca
     5881 ctacctgatc agatacggcg acccagctca cctgcacgag atcgctaaga acgaggccgt
     5941 ggtgaagttc gtgctgggac ggatcgccga tatccagaag aagcagggcc agaacggaaa
     6001 gaaccagatc gaccgctact acgagacctg catcggcaag gataagggaa agtccgtgtc
     6061 tgagaaggtg gacgctctga ccaagatcat cacaggcatg aactacgacc agttcgataa
     6121 gaagagatct gtgatcgagg acaccggaag ggagaacgcc gagagagaga agtttaagaa
     6181 gatcatcagc ctgtacctga cagtgatcta ccacatcctg aagaacatcg tgaacatcaa
     6241 cgctagatac gtgatcggct tccactgcgt ggagcgcgat gcccagctgt acaaggagaa
     6301 gggatacgac atcaacctga agaagctgga ggagaagggc tttagctccg tgaccaagct
     6361 gtgcgctgga atcgacgaga cagcccccga caagaggaag gatgtggaga aggagatggc
     6421 cgagagagct aaggagagca tcgactccct ggagtctgct aaccctaagc tgtacgccaa
     6481 ctacatcaag tactccgatg agaagaaggc cgaggagttc accaggcaga tcaacagaga
     6541 gaaggccaag accgctctga acgcctacct gaggaacaca aagtggaacg tgatcatccg
     6601 ggaggacctg ctgcgcatcg ataacaagac ctgtacactg ttcgctaaca aggctgtggc
     6661 cctggaggtg gctcgctacg tgcacgccta catcaacgac atcgccgagg tgaactccta
     6721 ctttcagctg taccactaca tcatgcagag gatcatcatg aacgagagat acgagaagtc
     6781 tagcggcaag gtgtctgagt acttcgacgc cgtgaacgat gagaagaagt acaacgatag
     6841 actgctgaag ctgctgtgcg tgcctttcgg atactgtatc ccacggttta agaacctgag
     6901 catcgaggcc ctgttcgacc gcaacgaggc tgccaagttt gataaggaga agaagaaggt
     6961 gagcggcaac tccggttcta agcgcaccgc cgatggatcc gagttcgaat cccctaaaaa
     7021 gaaaagaaag gtgggatccg actacaaaga ccatgacggt gattataaag atcatgatat
     7081 cgattacaag gatgacgatg acaagggatc caaaagaact ggaagcggag ctactaactt
     7141 cagcctgctg aagcaggctg gagacgtgga ggagaaccct ggacctgtta acatggtgag
     7201 caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat
     7261 ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
     7321 cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg
     7381 ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga
     7441 catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa
     7501 cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt
     7561 catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa
     7621 gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa
     7681 gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa
     7741 gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa
     7801 gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga
     7861 gggccgccac tccaccggcg gcatggacga gctgtacaag taagctcgag cctagagggc
     7921 ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     7981 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     8041 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     8101 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
     8161 tgggctctat ggtaccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     8221 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     8281 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     8341 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     8401 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     8461 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     8521 ccacagaatc aggggataac gcaggaaaga acatg
//
