LOCUS       Exported                6379 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  gRNA vector for AsCpf1.
ACCESSION   .
VERSION     .
KEYWORDS    pCFD7
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6379)
  AUTHORS   Port F, Bullock SL
  TITLE     Augmenting CRISPR applications in Drosophila with tRNA-flanked 
            sgRNAs.
  JOURNAL   Nat Methods. 2016 Oct;13(10):852-4. doi: 10.1038/nmeth.3972. Epub 
            2016 Sep 5.
  PUBMED    27595403
REFERENCE   2  (bases 1 to 6379)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6379
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        7..427
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     primer_bind     complement(956..976)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(956..974)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(995..1011)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(995..1011)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1008..1030)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1019..1035
                     /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(1043..1073)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1088..1109
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1226..1243)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1397..1985)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1477..1496)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2156..3016)
                     /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     2779..2798
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3017..3121)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3147..3602)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3284..3305)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3496..3515
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3729..3751
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3743..3760
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3744..3760
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3767..3786
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3767..3785
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(join(3977..4094,4171..4264,4356..4962,5017..5150,
                     5211..5371,5445..5470))
                     /codon_start=1
                     /product="tryptophan oxygenase"
                     /label=vermilion
                     /note="selectable marker, required to synthesize the brown 
                     eye pigment in Drosophila"
                     /translation="MSCPYAGNGNDHDDSAVPLTTEVGKIYGEYLMLDKLLDAQCMLSE
                     EDKRPVHDEHLFIITHQAYELWFKQIIFEFDSIRDMLDAEVIDETKTLEIVKRLNRVVL
                     ILKLLVDQVPILETMTPLDFMDFRKYLAPASGFQSLQFRLIENKLGVLTEQRVRYNQKY
                     SDVFSDEEARNSIRNSEKDPSLLELVQRWLERTPGLEESGFNFWAKFQESVDRFLEAQV
                     QSAMEEPVEKAKNYRLMDIEKRREVYRSIFDPAVHDALVRRGDRRFSHRALQGAIMITF
                     YRDEPRFSQPHQLLTLLMDIDSLITKWRYNHVIMVQRMIGSQQLGTGGSSGYQYLRSTL
                     SDRYKVFLDLFNLSTFLIPREAIPPLDETIRKKLINKSV"
     protein_bind    5785..5854
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
ORIGIN
        1 gaattctttt ttgctcacct gtgattgctc ctactcaaat acaaaaacat caaattttct
       61 gtcaataaag catatttatt tatatttatt ttacaggaaa gaattccttt taaagtgtat
      121 tttaacctat aatgaaaaac gattaaaaaa aatacataaa ataattcgaa aatttttgaa
      181 tagcccaggt tgataaaaat tcatttcata cgttttataa cttatgcccc taagtatttt
      241 ttgaccatag tgtttcaatt ctacattaat tttacagagt agaatgaaac gccacctact
      301 cagccaagag gcgaaaaggt tagctcgcca agcagagagg gcgccagtgc tcactacttt
      361 ttataattct caacttcttt ttccagactc agttcgtata tatagaccta ttttcaattt
      421 aacgtcgggg ctttgagtgt gtgtagacat caagcatcgg tggttcagtg gtagaatgct
      481 cgcctgccac gcgggcggcc cgggttcgat tcccggccga tgcataattt ctactcttgt
      541 agatgggtct tcgttatcag aagacctaac aaagcaccag tggtctagtg gtagaatagt
      601 accctgccac ggtacagacc cgggttcgat tcccggctgg tgcatttttt tgcctacctg
      661 gagcctgaga gttgttcaat aaaataaaaa tgtttcgttt ttttgctttc gccagtattt
      721 attatttttc atcaatatgt attcaatttg gtatgtattt agtaattgta atatatagac
      781 aatggttttc cgttgacgta catacatctg acgtgtgttt atttagacat aatagttatg
      841 ttttcacatc tttttaatgt tcgcttaatg cgtatgcatt ctagacaatt gtgctcggca
      901 acagtatatt tgtggtgtgc caaccaacaa cctgcaggag ctccagcttt tgttcccttt
      961 agtgagggtt aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     1021 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     1081 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     1141 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     1201 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     1261 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     1321 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     1381 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     1441 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     1501 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     1561 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     1621 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     1681 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     1741 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     1801 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     1861 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     1921 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     1981 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     2041 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     2101 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     2161 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     2221 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     2281 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     2341 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     2401 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     2461 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     2521 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     2581 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     2641 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     2701 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     2761 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     2821 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     2881 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     2941 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     3001 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     3061 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     3121 gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc
     3181 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     3241 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     3301 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     3361 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     3421 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     3481 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     3541 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     3601 gtcccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
     3661 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
     3721 agggttttcc cagtcacgac gttgtaaaac gacggccagt gaattgtaat acgactcact
     3781 atagggcgaa ttgggtacaa gcttggattt attttgttat gttatatgta ttatatgtca
     3841 gacataaaga aaaggaacac atcaaatgtg ataacaaaga ctaaacaagt aattttatta
     3901 caccaaaacg acaaaacagt aggcagaaca aacaacgcat agccaaacat tgacgaattg
     3961 gataccctgc cgattgtcag acacttttgt tgatcagttt cttgcgaatg gtctcgtcca
     4021 gcggtggaat cgcctcgcgg ggaatcagaa aagtggacag attgaacaga tccagaaaca
     4081 ccttgtaccg atcactgaaa ccaaaaaaaa acaaagggag aacagtttga gttcattgat
     4141 ccccgatata atcacatctg cgatgatcac ctgagagtgg agcgcagata ttgatatcca
     4201 gacgagccac cagtgcccaa ctgttgggat ccaatcatgc gttgcaccat gatcacgtga
     4261 ttgtctgcgg cgggaataga aagtatttgg ttaggaaaac cagtcttaaa cataagatat
     4321 atttataaaa gagtatcaaa gaatgcaata cttacatctc cacttggtta ttaacgagtc
     4381 gatgtccatg agcagggtga gcaactggtg tggttggctg aacctgggtt catccctata
     4441 gaaggtgatc atgatggctc cctgaagggc acgatggcta aaccggcgat ccccacgacg
     4501 caccagtgca tcgtgcactg ccggatcaaa gatggagcga tacacctcgc gtcgcttctc
     4561 aatgtccatg aggcggtagt ttttcgcctt ctccacgggc tcctccatgg cgctctgtac
     4621 ctgcgcctcc aggaatcgat cgacgctctc ctgaaacttg gcccagaagt tgaagccact
     4681 ctcctccagt ccgggcgtcc tctccagcca tcgctgcact agctccagta gcgagggatc
     4741 tttctccgag ttgcgaatcg agttccgcgc ctcctcgtcg ctaaagacat ccgagtactt
     4801 ctggttgtat ctcacccgct gctctgtcag aactcccagc ttgttctcga tcaaacggaa
     4861 ctgcagcgac tgaaaaccag atgcgggtgc caggtacttg cggaagtcca tgaagtctag
     4921 cggggtcatg gtctccagaa tgggcacttg gtccaccagg agctgtacaa aggaagttat
     4981 aaacggattt tggtaagaga ttcagaaagc actcactttt agaatcagaa ccactcggtt
     5041 cagtcgcttg acaatctcca gcgtcttggt ttcatcgatg acctctgcat ccaacatgtc
     5101 tcgtatggag tcgaactcaa agatgatctg cttgaaccaa agctcgtagg ctgtggcgaa
     5161 ggtacttaaa tgccattgag tgttgtcatc aaagttgtaa acctactcac cctggtgcgt
     5221 gatgatgaac agatgctcat cgtgcacggg tcgcttgtcc tcctcggaca gcatacactg
     5281 ggcatccagc agtttgtcca gcatcagata ctctccatag attttgccca cttccgtggt
     5341 taatggcacc gccgaatcat cgtgatcgtt tctgtatggg tttgaattga atcgcagaac
     5401 tgaagatcga ttggcattcc tggacagcac gtgctggtgc tcacccgttt cctgcatagg
     5461 gacagctcat ggtgcacagc tcagatcaga tcgtgactcc tcgagcggcg gatgctggcg
     5521 aactgatctc cgccagcgga ccggagatga gaccccagcg aaccgataac agagcgagag
     5581 agctccagtt ccgactgatt gcacagtcgg tgatctgggc gatgggcact gccagatagg
     5641 ctgggaatta tcaatcactt gaggtgaaag tgcggcgcac acaaatccaa gcttgatatc
     5701 atcgatctcg aggctgcatc caacgcgttg ggagctctcc ggatcaattc ggcttcaggt
     5761 accgtcgacg atgtaggtca cggtctcgaa gccgcggtgc gggtgccagg gcgtgccctt
     5821 gggctccccg ggcgcgtact ccacctcacc catctggtcc atcatgatga acgggtcgag
     5881 gtggcggtag ttgatcccgg cgaacgcgcg gcgcaccggg aagccctcgc cctcgaaacc
     5941 gctgggcgcg gtggtcacgg tgagcacggg acgtgcgacg gcgtcggctg gtgcggatac
     6001 gcggggcagc gtcagcgggt tctcgacggt cacggcgggc atgtcgacaa gccgaattga
     6061 tccactagaa ggcctaattc ggtacactag ttggccacgt aataagtgtg cgttgaattt
     6121 attcgcaaaa acattgcata ttttcggcaa agtaaaattt tgttgcatac cttatcaaaa
     6181 aataagtgct gcatactttt tagagaaacc aaataatttt ttattgcata cccgttttta
     6241 ataaaataca ttgcataccc tcttttaata aagaatattg catacgttga cgaaacaaat
     6301 tttcgttgca tacccaataa aagattatta tattgcatac cttttcttgc cataccattt
     6361 agccgatcaa ttgagatct
//
