LOCUS       sequence-337340-        7259 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression of two gRNAs from Drosophila U6:1 and U6:3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7259)
  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 7259)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl 
            Acad Sci U S A. 2014 Jul 7. pii: 201405500."
COMMENT     Sequence Label: pCFD4-U6:1_U6:3tandemgRNAs
FEATURES             Location/Qualifiers
     source          1..7259
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..457
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(88..107)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     298..319
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     583..605
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     597..614
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     598..614
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     624..643
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        624..642
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(join(834..951,1028..1121,1213..1819,1874..2007,
                     2068..2228,2302..2327))
                     /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    2642..2711
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     protein_bind    2953..3338
                     /label=gypsy insulator
                     /bound_moiety="Su(Hw), CP190, andMod(mdg4)67.2"
                     /note="chromatin insulator from Drosophila"
     promoter        3352..4148
                     /label=dU6-1 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-1 
                     snRNA (Port et al., 2014)"
     misc_RNA        4167..4242
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        4243..4663
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_RNA        4682..4757
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(5068..5088)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5068..5086)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5107..5123)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5107..5123)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5120..5142)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5131..5147
                     /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(join(5155..5161,5162..5179,5180..5185))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5200..5221
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5338..5355)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5509..6097)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5589..5608)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6268..7059,7060..7128))
                     /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     6891..6910
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7129..7233)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
       61 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
      121 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
      181 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
      241 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
      301 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
      361 aagggatttt gccgatttcg gcctattggt taaaaaaatg agctgattta acaaaaattt
      421 aacgcgaatt ttaacaaaat attaacgctt acaatttcca ttcgccattc aggctgcgca
      481 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
      541 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
      601 aaacgacggc cagtgagcgc gcgtaatacg actcactata gggcgaattg ggtacaagct
      661 tggatttatt ttgttatgtt atatgtatta tatgtcagac ataaagaaaa ggaacacatc
      721 aaatgtgata acaaagacta aacaagtaat tttattacac caaaacgaca aaacagtagg
      781 cagaacaaac aacgcatagc caaacattga cgaattggat accctgccga ttgtcagaca
      841 cttttgttga tcagtttctt gcgaatggtc tcgtccagcg gtggaatcgc ctcgcgggga
      901 atcagaaaag tggacagatt gaacagatcc agaaacacct tgtaccgatc actgaaacca
      961 aaaaaaaaca aagggagaac agtttgagtt cattgatccc cgatataatc acatctgcga
     1021 tgatcacctg agagtggagc gcagatattg atatccagac gagccaccag tgcccaactg
     1081 ttgggatcca atcatgcgtt gcaccatgat cacgtgattg tctgcggcgg gaatagaaag
     1141 tatttggtta ggaaaaccag tcttaaacat aagatatatt tataaaagag tatcaaagaa
     1201 tgcaatactt acatctccac ttggttatta acgagtcgat gtccatgagc agggtgagca
     1261 actggtgtgg ttggctgaac ctgggttcat ccctatagaa ggtgatcatg atggctccct
     1321 gaagggcacg atggctaaac cggcgatccc cacgacgcac cagtgcatcg tgcactgccg
     1381 gatcaaagat ggagcgatac acctcgcgtc gcttctcaat gtccatgagg cggtagtttt
     1441 tcgccttctc cacgggctcc tccatggcgc tctgtacctg cgcctccagg aatcgatcga
     1501 cgctctcctg aaacttggcc cagaagttga agccactctc ctccagtccg ggcgtcctct
     1561 ccagccatcg ctgcactagc tccagtagcg agggatcttt ctccgagttg cgaatcgagt
     1621 tccgcgcctc ctcgtcgcta aagacatccg agtacttctg gttgtatctc acccgctgct
     1681 ctgtcagaac tcccagcttg ttctcgatca aacggaactg cagcgactga aaaccagatg
     1741 cgggtgccag gtacttgcgg aagtccatga agtctagcgg ggtcatggtc tccagaatgg
     1801 gcacttggtc caccaggagc tgtacaaagg aagttataaa cggattttgg taagagattc
     1861 agaaagcact cacttttaga atcagaacca ctcggttcag tcgcttgaca atctccagcg
     1921 tcttggtttc atcgatgacc tctgcatcca acatgtctcg tatggagtcg aactcaaaga
     1981 tgatctgctt gaaccaaagc tcgtaggctg tggcgaaggt acttaaatgc cattgagtgt
     2041 tgtcatcaaa gttgtaaacc tactcaccct ggtgcgtgat gatgaacaga tgctcatcgt
     2101 gcacgggtcg cttgtcctcc tcggacagca tacactgggc atccagcagt ttgtccagca
     2161 tcagatactc tccatagatt ttgcccactt ccgtggttaa tggcaccgcc gaatcatcgt
     2221 gatcgtttct gtatgggttt gaattgaatc gcagaactga agatcgattg gcattcctgg
     2281 acagcacgtg ctggtgctca cccgtttcct gcatagggac agctcatggt gcacagctca
     2341 gatcagatcg tgactcctcg agcggcggat gctggcgaac tgatctccgc cagcggaccg
     2401 gagatgagac cccagcgaac cgataacaga gcgagagagc tccagttccg actgattgca
     2461 cagtcggtga tctgggcgat gggcactgcc agataggctg ggaattatca atcacttgag
     2521 gtgaaagtgc ggcgcacaca aatccaagct tgatatcatc gatctcgagg ctgcatccaa
     2581 cgcgttggga gctctccgga tcaattcggc ttcaggtacc gtcgacgatg taggtcacgg
     2641 tctcgaagcc gcggtgcggg tgccagggcg tgcccttggg ctccccgggc gcgtactcca
     2701 cctcacccat ctggtccatc atgatgaacg ggtcgaggtg gcggtagttg atcccggcga
     2761 acgcgcggcg caccgggaag ccctcgccct cgaaaccgct gggcgcggtg gtcacggtga
     2821 gcacgggacg tgcgacggcg tcggctggtg cggatacgcg gggcagcgtc agcgggttct
     2881 cgacggtcac ggcgggcatg tcgacaagcc gaattgatcc actagaaggc ctaattcggt
     2941 acactagttg gccacgtaat aagtgtgcgt tgaatttatt cgcaaaaaca ttgcatattt
     3001 tcggcaaagt aaaattttgt tgcatacctt atcaaaaaat aagtgctgca tactttttag
     3061 agaaaccaaa taatttttta ttgcataccc gtttttaata aaatacattg cataccctct
     3121 tttaataaaa aatattgcat actttgacga aacaaatttt cgttgcatac ccaataaaag
     3181 attattatat tgcatacccg tttttaataa aatacattgc ataccctctt ttaataaaga
     3241 atattgcata cgttgacgaa acaaattttc gttgcatacc caataaaaga ttattatatt
     3301 gcataccttt tcttgccata ccatttagcc gatcaattga gatctgaatt cattttcaac
     3361 gtcctcgata gtatagtggt tagtatcccc gcctgtcacg cgggagaccg gggttcaatt
     3421 ccccgtcggg gagaatctgt gattcttttt ttttttcttt tactttgtta tataaacaat
     3481 ttttgtttta attgaatcta atttgccatt gcttttagga atctcaggca tccagcaagc
     3541 gtttgtccgc cgaatcgccc atcagtgaag aagatcctgt ggcggctacg aaaatctccc
     3601 cggccatgtc ggcctccacc tccagcgaaa aacccatcag cgagctggcc acctctgtgc
     3661 tgacccaccg ctttccagac tccacctcct cacccggcga acatggcctt ggacgaatgc
     3721 agttgtcgat ccgctacagc gcccagcgtc aaaaactaga cgtgaccata cacaaaatcc
     3781 agaagatacc acttcgcgat cccagcaata tccccgatcc gtatgttaag ctgtatctgt
     3841 tgcctggacg caccaaggag tcgaaacgca agacgagcgt gatcaaggac aactgcaacc
     3901 ccgtctacga tgcatccttt gagtacctga tttccattgc cgaactcagg cagacggaac
     3961 tggaggtgac ggtgtgcacc caaaagggat tcctatccgg cggtagtccc atcattggca
     4021 tggtaggtac ccgaaagcaa ccccttagtt acagacacag cgcgtacgtc cttcgcatcc
     4081 ttatgattcc caagtacata ttctgcaaga gtacagtata tataggaaag atatccgggt
     4141 gaacttcggg gtcttcgaga agacctgttt tagagctaga aatagcaagt taaaataagg
     4201 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttgc tcacctgtga
     4261 ttgctcctac tcaaatacaa aaacatcaaa ttttctgtca ataaagcata tttatttata
     4321 tttattttac aggaaagaat tccttttaaa gtgtatttta acctataatg aaaaacgatt
     4381 aaaaaaaata cataaaataa ttcgaaaatt tttgaatagc ccaggttgat aaaaattcat
     4441 ttcatacgtt ttataactta tgcccctaag tattttttga ccatagtgtt tcaattctac
     4501 attaatttta cagagtagaa tgaaacgcca cctactcagc caagaggcga aaaggttagc
     4561 tcgccaagca gagagggcgc cagtgctcac tactttttat aattctcaac ttctttttcc
     4621 agactcagtt cgtatatata gacctatttt caatttaacg tcggggtctt cgagaagacc
     4681 tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
     4741 tggcaccgag tcggtgcttt tttgcctacc tggagcctga gagttgttca ataaaataaa
     4801 aatgtttcgt ttttttgctt tcgccagtat ttattatttt tcatcaatat gtattcaatt
     4861 tggtatgtat ttagtaattg taatatatag acaatggttt tccgttgacg tacatacatc
     4921 tgacgtgtgt ttatttagac ataatagtta tgttttcaca tctttttaat gttcgcttaa
     4981 tgcgtatgca ttctagacaa ttgtgctcgg caacagtata tttgtggtgt gccaaccaac
     5041 aacctgcagg agctccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta
     5101 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     5161 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     5221 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     5281 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     5341 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     5401 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     5461 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     5521 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     5581 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     5641 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     5701 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     5761 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     5821 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     5881 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     5941 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     6001 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     6061 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     6121 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     6181 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     6241 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     6301 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     6361 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     6421 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     6481 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     6541 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     6601 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     6661 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     6721 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     6781 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     6841 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     6901 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     6961 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     7021 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     7081 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     7141 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     7201 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctg
//
