LOCUS       Exported                6624 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  expression of gRNA under control of the Drosophila U6:1 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    pCFD1-dU6:1gRNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6624)
  AUTHORS   Port F, Chen HM, Lee T, Bullock SL
  TITLE     Optimized CRISPR/Cas tools for efficient germline and somatic genome
            engineering in Drosophila.
  JOURNAL   Proc Natl Acad Sci U S A. 2014 Jul 7. pii: 201405500.
  PUBMED    25002478
REFERENCE   2  (bases 1 to 6624)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6624
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        7..803
                     /label=dU6-1 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-1 
                     snRNA (Port et al., 2014)"
     misc_RNA        822..897
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1208..1228)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1208..1226)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1247..1263)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1247..1263)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1260..1282)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1271..1287
                     /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(1295..1325)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1340..1361
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1478..1495)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1649..2237)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1729..1748)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2408..3268)
                     /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     3031..3050
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3269..3373)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3399..3854)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3536..3557)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3748..3767
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3981..4003
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3995..4012
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3996..4012
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4019..4038
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4019..4037
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(join(4229..4346,4423..4516,4608..5214,5269..5402,
                     5463..5623,5697..5722))
                     /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    6037..6106
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
ORIGIN
        1 gaattcattt tcaacgtcct cgatagtata gtggttagta tccccgcctg tcacgcggga
       61 gaccggggtt caattccccg tcggggagaa tctgtgattc tttttttttt tcttttactt
      121 tgttatataa acaatttttg ttttaattga atctaatttg ccattgcttt taggaatctc
      181 aggcatccag caagcgtttg tccgccgaat cgcccatcag tgaagaagat cctgtggcgg
      241 ctacgaaaat ctccccggcc atgtcggcct ccacctccag cgaaaaaccc atcagcgagc
      301 tggccacctc tgtgctgacc caccgctttc cagactccac ctcctcaccc ggcgaacatg
      361 gccttggacg aatgcagttg tcgatccgct acagcgccca gcgtcaaaaa ctagacgtga
      421 ccatacacaa aatccagaag ataccacttc gcgatcccag caatatcccc gatccgtatg
      481 ttaagctgta tctgttgcct ggacgcacca aggagtcgaa acgcaagacg agcgtgatca
      541 aggacaactg caaccccgtc tacgatgcat cctttgagta cctgatttcc attgccgaac
      601 tcaggcagac ggaactggag gtgacggtgt gcacccaaaa gggattccta tccggcggta
      661 gtcccatcat tggcatggta ggtacccgaa agcaacccct tagttacaga cacagcgcgt
      721 acgtccttcg catccttatg attcccaagt acatattctg caagagtaca gtatatatag
      781 gaaagatatc cgggtgaact tcggggtctt cgagaagacc tgttttagag ctagaaatag
      841 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
      901 tttgcctacc tggagcctga gagttgttca ataaaataaa aatgtttcgt ttttttgctt
      961 tcgccagtat ttattatttt tcatcaatat gtattcaatt tggtatgtat ttagtaattg
     1021 taatatatag acaatggttt tccgttgacg tacatacatc tgacgtgtgt ttatttagac
     1081 ataatagtta tgttttcaca tctttttaat gttcgcttaa tgcgtatgca ttctagacaa
     1141 ttgtgctcgg caacagtata tttgtggtgt gccaaccaac aacctgcagg agctccagct
     1201 tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc
     1261 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     1321 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     1381 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     1441 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     1501 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     1561 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     1621 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     1681 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     1741 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     1801 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     1861 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     1921 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     1981 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     2041 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     2101 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     2161 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     2221 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     2281 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     2341 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     2401 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     2461 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     2521 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     2581 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     2641 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     2701 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     2761 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     2821 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     2881 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     2941 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     3001 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     3061 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     3121 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     3181 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     3241 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     3301 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     3361 taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt
     3421 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     3481 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     3541 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     3601 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     3661 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     3721 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     3781 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     3841 gctacagggc gcgtcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt
     3901 gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag
     3961 ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgaattgta
     4021 atacgactca ctatagggcg aattgggtac aagcttggat ttattttgtt atgttatatg
     4081 tattatatgt cagacataaa gaaaaggaac acatcaaatg tgataacaaa gactaaacaa
     4141 gtaattttat tacaccaaaa cgacaaaaca gtaggcagaa caaacaacgc atagccaaac
     4201 attgacgaat tggataccct gccgattgtc agacactttt gttgatcagt ttcttgcgaa
     4261 tggtctcgtc cagcggtgga atcgcctcgc ggggaatcag aaaagtggac agattgaaca
     4321 gatccagaaa caccttgtac cgatcactga aaccaaaaaa aaacaaaggg agaacagttt
     4381 gagttcattg atccccgata taatcacatc tgcgatgatc acctgagagt ggagcgcaga
     4441 tattgatatc cagacgagcc accagtgccc aactgttggg atccaatcat gcgttgcacc
     4501 atgatcacgt gattgtctgc ggcgggaata gaaagtattt ggttaggaaa accagtctta
     4561 aacataagat atatttataa aagagtatca aagaatgcaa tacttacatc tccacttggt
     4621 tattaacgag tcgatgtcca tgagcagggt gagcaactgg tgtggttggc tgaacctggg
     4681 ttcatcccta tagaaggtga tcatgatggc tccctgaagg gcacgatggc taaaccggcg
     4741 atccccacga cgcaccagtg catcgtgcac tgccggatca aagatggagc gatacacctc
     4801 gcgtcgcttc tcaatgtcca tgaggcggta gtttttcgcc ttctccacgg gctcctccat
     4861 ggcgctctgt acctgcgcct ccaggaatcg atcgacgctc tcctgaaact tggcccagaa
     4921 gttgaagcca ctctcctcca gtccgggcgt cctctccagc catcgctgca ctagctccag
     4981 tagcgaggga tctttctccg agttgcgaat cgagttccgc gcctcctcgt cgctaaagac
     5041 atccgagtac ttctggttgt atctcacccg ctgctctgtc agaactccca gcttgttctc
     5101 gatcaaacgg aactgcagcg actgaaaacc agatgcgggt gccaggtact tgcggaagtc
     5161 catgaagtct agcggggtca tggtctccag aatgggcact tggtccacca ggagctgtac
     5221 aaaggaagtt ataaacggat tttggtaaga gattcagaaa gcactcactt ttagaatcag
     5281 aaccactcgg ttcagtcgct tgacaatctc cagcgtcttg gtttcatcga tgacctctgc
     5341 atccaacatg tctcgtatgg agtcgaactc aaagatgatc tgcttgaacc aaagctcgta
     5401 ggctgtggcg aaggtactta aatgccattg agtgttgtca tcaaagttgt aaacctactc
     5461 accctggtgc gtgatgatga acagatgctc atcgtgcacg ggtcgcttgt cctcctcgga
     5521 cagcatacac tgggcatcca gcagtttgtc cagcatcaga tactctccat agattttgcc
     5581 cacttccgtg gttaatggca ccgccgaatc atcgtgatcg tttctgtatg ggtttgaatt
     5641 gaatcgcaga actgaagatc gattggcatt cctggacagc acgtgctggt gctcacccgt
     5701 ttcctgcata gggacagctc atggtgcaca gctcagatca gatcgtgact cctcgagcgg
     5761 cggatgctgg cgaactgatc tccgccagcg gaccggagat gagaccccag cgaaccgata
     5821 acagagcgag agagctccag ttccgactga ttgcacagtc ggtgatctgg gcgatgggca
     5881 ctgccagata ggctgggaat tatcaatcac ttgaggtgaa agtgcggcgc acacaaatcc
     5941 aagcttgata tcatcgatct cgaggctgca tccaacgcgt tgggagctct ccggatcaat
     6001 tcggcttcag gtaccgtcga cgatgtaggt cacggtctcg aagccgcggt gcgggtgcca
     6061 gggcgtgccc ttgggctccc cgggcgcgta ctccacctca cccatctggt ccatcatgat
     6121 gaacgggtcg aggtggcggt agttgatccc ggcgaacgcg cggcgcaccg ggaagccctc
     6181 gccctcgaaa ccgctgggcg cggtggtcac ggtgagcacg ggacgtgcga cggcgtcggc
     6241 tgggtgcgga tacgcggggc agcgtcagcg ggttctcgac ggtcacggcg ggcatgtcga
     6301 caagccgaat tgatccacta gaaggcctaa ttcggtacac tagttggcca cgtaataagt
     6361 gtgcgttgaa tttattcgca aaaacattgc atattttcgg caaagtaaaa ttttgttgca
     6421 taccttatca aaaaataagt gctgcatact ttttagagaa accaaataat tttttattgc
     6481 atacccgttt ttaataaaat acattgcata ccctctttta ataaaaaata ttgcatactt
     6541 tgacgaaaca aattttcgtt gcatacccaa taaaagatta ttatattgca tacccgtttt
     6601 taataaaata cattgcatag atct
//
