LOCUS       62056_2380        8560 bp DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8560)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..8560
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        231..1409
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation
                     factor EF-1-alpha"
     CDS             1431..1451
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     CDS             1461..4358
                     /codon_start=1
                     /label=RfxCas13d
                     /note="RNA-targeting Type VI CRISPR protein from
                     Ruminococcus flavefaciens strain XPD3002 (Konermann et al.,
                     2018)"
                     /translation="IEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDSI
                     RSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEK
                     RYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIGFGKFSTVY
                     TYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYIIN
                     YGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNE
                     LTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRKD
                     MSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFVI
                     NLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAG
                     RDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFF
                     KDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDEN
                     GNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQK
                     KQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGREN
                     AEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEE
                     KGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKKA
                     EEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVHA
                     YINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCVP
                     FGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS"
     CDS             4368..4394
                     /codon_start=1
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
                     /translation="YPYDVPDYA"
     CDS             4395..4415
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     CDS             4482..5198
                     /codon_start=1
                     /label=EGFP
                     /note="enhanced GFP"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     polyA_signal    5217..5351
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        5358..5598
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        5608..5637
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d
                     gRNA (Konermann et al., 2018)"
     misc_RNA        5668..5703
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     repeat_region   5823..5963
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      6038..6493
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        6775..6879
                     /label=AmpR promoter
     CDS             6880..7737
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      7911..8499
                     /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 gcggccgcac gcgtaagctt tgcaaagatg gataaagttt taaacagaga
      181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
      481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
      541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
      601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
      661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
      721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
      781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
      841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
      901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
      961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1381 gtttttttct tccatttcag gtgtcgtgag gtacgaccgg tgccaccatg cctaaaaaga
     1441 aaagaaaggt gggttctggt atcgaaaaaa aaaagtcctt cgccaagggc atgggcgtga
     1501 agtccacact cgtgtccggc tccaaagtgt acatgacaac cttcgccgaa ggcagcgacg
     1561 ccaggctgga aaagatcgtg gagggcgaca gcatcaggag cgtgaatgag ggcgaggcct
     1621 tcagcgctga aatggccgat aaaaacgccg gctataagat cggcaacgcc aaattcagcc
     1681 atcctaaggg ctacgccgtg gtggctaaca accctctgta tacaggaccc gtccagcagg
     1741 atatgctcgg cctgaaggaa actctggaaa agaggtactt cggcgagagc gctgatggca
     1801 atgacaatat ttgtatccag gtgatccata acatcctgga cattgaaaaa atcctcgccg
     1861 aatacattac caacgccgcc tacgccgtca acaatatctc cggcctggat aaggacatta
     1921 ttggattcgg caagttctcc acagtgtata cctacgacga attcaaagac cccgagcacc
     1981 atagggccgc tttcaacaat aacgataagc tcatcaacgc catcaaggcc cagtatgacg
     2041 agttcgacaa cttcctcgat aaccccagac tcggctattt cggccaggcc tttttcagca
     2101 aggagggcag aaattacatc atcaattacg gcaacgaatg ctatgacatt ctggccctcc
     2161 tgagcggact gaggcactgg gtggtccata acaacgaaga agagtccagg atctccagga
     2221 cctggctcta caacctcgat aagaacctcg acaacgaata catctccacc ctcaactacc
     2281 tctacgacag gatcaccaat gagctgacca actccttctc caagaactcc gccgccaacg
     2341 tgaactatat tgccgaaact ctgggaatca accctgccga attcgccgaa caatatttca
     2401 gattcagcat tatgaaagag cagaaaaacc tcggattcaa tatcaccaag ctcagggaag
     2461 tgatgctgga caggaaggat atgtccgaga tcaggaaaaa tcataaggtg ttcgactcca
     2521 tcaggaccaa ggtctacacc atgatggact ttgtgattta taggtattac atcgaagagg
     2581 atgccaaggt ggctgccgcc aataagtccc tccccgataa tgagaagtcc ctgagcgaga
     2641 aggatatctt tgtgattaac ctgaggggct ccttcaacga cgaccagaag gatgccctct
     2701 actacgatga agctaataga atttggagaa agctcgaaaa tatcatgcac aacatcaagg
     2761 aatttagggg aaacaagaca agagagtata agaagaagga cgcccctaga ctgcccagaa
     2821 tcctgcccgc tggccgtgat gtttccgcct tcagcaaact catgtatgcc ctgaccatgt
     2881 tcctggatgg caaggagatc aacgacctcc tgaccaccct gattaataaa ttcgataaca
     2941 tccagagctt cctgaaggtg atgcctctca tcggagtcaa cgctaagttc gtggaggaat
     3001 acgccttttt caaagactcc gccaagatcg ccgatgagct gaggctgatc aagtccttcg
     3061 ctagaatggg agaacctatt gccgatgcca ggagggccat gtatatcgac gccatccgta
     3121 ttttaggaac caacctgtcc tatgatgagc tcaaggccct cgccgacacc ttttccctgg
     3181 acgagaacgg aaacaagctc aagaaaggca agcacggcat gagaaatttc attattaata
     3241 acgtgatcag caataaaagg ttccactacc tgatcagata cggtgatcct gcccacctcc
     3301 atgagatcgc caaaaacgag gccgtggtga agttcgtgct cggcaggatc gctgacatcc
     3361 agaaaaaaca gggccagaac ggcaagaacc agatcgacag gtactacgaa acttgtatcg
     3421 gaaaggataa gggcaagagc gtgagcgaaa aggtggacgc tctcacaaag atcatcaccg
     3481 gaatgaacta cgaccaattc gacaagaaaa ggagcgtcat tgaggacacc ggcagggaaa
     3541 acgccgagag ggagaagttt aaaaagatca tcagcctgta cctcaccgtg atctaccaca
     3601 tcctcaagaa tattgtcaat atcaacgcca ggtacgtcat cggattccat tgcgtcgagc
     3661 gtgatgctca actgtacaag gagaaaggct acgacatcaa tctcaagaaa ctggaagaga
     3721 agggattcag ctccgtcacc aagctctgcg ctggcattga tgaaactgcc cccgataaga
     3781 gaaaggacgt ggaaaaggag atggctgaaa gagccaagga gagcattgac agcctcgaga
     3841 gcgccaaccc caagctgtat gccaattaca tcaaatacag cgacgagaag aaagccgagg
     3901 agttcaccag gcagattaac agggagaagg ccaaaaccgc cctgaacgcc tacctgagga
     3961 acaccaagtg gaatgtgatc atcagggagg acctcctgag aattgacaac aagacatgta
     4021 ccctgttcag aaacaaggcc gtccacctgg aagtggccag gtatgtccac gcctatatca
     4081 acgacattgc cgaggtcaat tcctacttcc aactgtacca ttacatcatg cagagaatta
     4141 tcatgaatga gaggtacgag aaaagcagcg gaaaggtgtc cgagtacttc gacgctgtga
     4201 atgacgagaa gaagtacaac gataggctcc tgaaactgct gtgtgtgcct ttcggctact
     4261 gtatccccag gtttaagaac ctgagcatcg aggccctgtt cgataggaac gaggccgcca
     4321 agttcgacaa ggagaaaaag aaggtgtccg gcaattccgg ttctggttac ccatacgacg
     4381 taccagatta cgctcccaag aagaagcgca aggtgggatc cggcgcaaca aacttctctc
     4441 tgctgaaaca agccggagat gtcgaagaga atcctggacc gatggtgtct aagggcgaag
     4501 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     4561 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     4621 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     4681 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     4741 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     4801 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     4861 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     4921 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     4981 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     5041 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     5101 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     5161 ccgccgggat cactctcggc atggacgagc tgtacaagta aggataaaga tcctaaaact
     5221 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     5281 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     5341 atgtctggat cgcggccgag ggcctatttc ccatgattcc ttcatatttg catatacgat
     5401 acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag atattagtac
     5461 aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt
     5521 ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat
     5581 atatcttgtg gaaaggacga aacaccgaac ccctaccaac tggtcggggt ttgaaacggg
     5641 gaagctacac tgttgtccaa gccatcacaa gtaaacccct accaactggt cggggtttga
     5701 aacacaggag ctggagatgc cccctgaggc acccaagtaa acccctacca actggtcggg
     5761 gtttgaaact ttttttgaat tctgatgcgg tggcggccgc cacgtgcgga ccgagcggcc
     5821 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     5881 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     5941 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg
     6001 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     6061 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg
     6121 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     6181 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     6241 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     6301 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     6361 acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc
     6421 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     6481 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     6541 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     6601 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     6661 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     6721 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     6781 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     6841 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     6901 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     6961 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     7021 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     7081 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     7141 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     7201 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     7261 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     7321 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     7381 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     7441 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     7501 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     7561 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     7621 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     7681 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     7741 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     7801 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     7861 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     7921 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     7981 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     8041 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     8101 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     8161 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     8221 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     8281 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     8341 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     8401 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     8461 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     8521 ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
