LOCUS       sequence-73454-e        6227 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression of gRNA under control of the Drosophila U6:2 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6227)
  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 6227)
  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: pCFD2-dU6:2gRNA
FEATURES             Location/Qualifiers
     source          1..6227
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..406
                     /label=dU6-2 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-2 
                     snRNA (Das et al., 1987)"
     misc_RNA        425..500
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(811..831)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(811..829)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(850..866)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(850..866)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(863..885)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    874..890
                     /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(898..904,905..922,923..928))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    943..964
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1081..1098)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1252..1840)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1332..1351)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2011..2802,2803..2871))
                     /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     2634..2653
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2872..2976)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3002..3457)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3139..3160)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3351..3370
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3584..3606
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3598..3615
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3599..3615
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3622..3641
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3622..3640
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(join(3832..3949,4026..4119,4211..4817,4872..5005,
                     5066..5226,5300..5325))
                     /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    5640..5709
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
ORIGIN
        1 gaattcgttc gacttgcagc ctgaaatacg gcacgagtag gaaaagccga gtcaaatgcc
       61 gaatgcagag tctcattaca gcacaatcaa ctcaagaaaa actcgacact tttttaccat
      121 ttgcacttaa atcctttttt attcgttatg tatacttttt ttggtcccta accaaaacaa
      181 aaccaaactc tcttagtcgt gcctctatat ttaaaactat caatttatta tagtcaataa
      241 atcgaactgt gttttcaaca aacgaacaat aggacacttt gattctaaag gaaattttga
      301 aaatcttaag cagagggttc ttaagaccat ttgccaattc ttataattct caactgctct
      361 ttcctgatgt tgatcattta tataggtatg ttttcctcaa tacttcgggt cttcgagaag
      421 acctgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
      481 aagtggcacc gagtcggtgc ttttttgcct acctggagcc tgagagttgt tcaataaaat
      541 aaaaatgttt cgtttttttg ctttcgccag tatttattat ttttcatcaa tatgtattca
      601 atttggtatg tatttagtaa ttgtaatata tagacaatgg ttttccgttg acgtacatac
      661 atctgacgtg tgtttattta gacataatag ttatgttttc acatcttttt aatgttcgct
      721 taatgcgtat gcattctaga caattgtgct cggcaacagt atatttgtgg tgtgccaacc
      781 aacaacctgc aggagctcca gcttttgttc cctttagtga gggttaattg cgcgcttggc
      841 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
      901 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
      961 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     1021 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     1081 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     1141 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     1201 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     1261 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     1321 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     1381 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     1441 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     1501 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     1561 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     1621 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     1681 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     1741 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     1801 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     1861 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     1921 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     1981 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     2041 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     2101 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     2161 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     2221 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     2281 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     2341 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     2401 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     2461 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     2521 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     2581 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     2641 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     2701 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     2761 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     2821 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     2881 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     2941 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     3001 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     3061 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     3121 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     3181 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     3241 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     3301 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     3361 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     3421 acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc
     3481 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg
     3541 ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt
     3601 aaaacgacgg ccagtgaatt gtaatacgac tcactatagg gcgaattggg tacaagcttg
     3661 gatttatttt gttatgttat atgtattata tgtcagacat aaagaaaagg aacacatcaa
     3721 atgtgataac aaagactaaa caagtaattt tattacacca aaacgacaaa acagtaggca
     3781 gaacaaacaa cgcatagcca aacattgacg aattggatac cctgccgatt gtcagacact
     3841 tttgttgatc agtttcttgc gaatggtctc gtccagcggt ggaatcgcct cgcggggaat
     3901 cagaaaagtg gacagattga acagatccag aaacaccttg taccgatcac tgaaaccaaa
     3961 aaaaaacaaa gggagaacag tttgagttca ttgatccccg atataatcac atctgcgatg
     4021 atcacctgag agtggagcgc agatattgat atccagacga gccaccagtg cccaactgtt
     4081 gggatccaat catgcgttgc accatgatca cgtgattgtc tgcggcggga atagaaagta
     4141 tttggttagg aaaaccagtc ttaaacataa gatatattta taaaagagta tcaaagaatg
     4201 caatacttac atctccactt ggttattaac gagtcgatgt ccatgagcag ggtgagcaac
     4261 tggtgtggtt ggctgaacct gggttcatcc ctatagaagg tgatcatgat ggctccctga
     4321 agggcacgat ggctaaaccg gcgatcccca cgacgcacca gtgcatcgtg cactgccgga
     4381 tcaaagatgg agcgatacac ctcgcgtcgc ttctcaatgt ccatgaggcg gtagtttttc
     4441 gccttctcca cgggctcctc catggcgctc tgtacctgcg cctccaggaa tcgatcgacg
     4501 ctctcctgaa acttggccca gaagttgaag ccactctcct ccagtccggg cgtcctctcc
     4561 agccatcgct gcactagctc cagtagcgag ggatctttct ccgagttgcg aatcgagttc
     4621 cgcgcctcct cgtcgctaaa gacatccgag tacttctggt tgtatctcac ccgctgctct
     4681 gtcagaactc ccagcttgtt ctcgatcaaa cggaactgca gcgactgaaa accagatgcg
     4741 ggtgccaggt acttgcggaa gtccatgaag tctagcgggg tcatggtctc cagaatgggc
     4801 acttggtcca ccaggagctg tacaaaggaa gttataaacg gattttggta agagattcag
     4861 aaagcactca cttttagaat cagaaccact cggttcagtc gcttgacaat ctccagcgtc
     4921 ttggtttcat cgatgacctc tgcatccaac atgtctcgta tggagtcgaa ctcaaagatg
     4981 atctgcttga accaaagctc gtaggctgtg gcgaaggtac ttaaatgcca ttgagtgttg
     5041 tcatcaaagt tgtaaaccta ctcaccctgg tgcgtgatga tgaacagatg ctcatcgtgc
     5101 acgggtcgct tgtcctcctc ggacagcata cactgggcat ccagcagttt gtccagcatc
     5161 agatactctc catagatttt gcccacttcc gtggttaatg gcaccgccga atcatcgtga
     5221 tcgtttctgt atgggtttga attgaatcgc agaactgaag atcgattggc attcctggac
     5281 agcacgtgct ggtgctcacc cgtttcctgc atagggacag ctcatggtgc acagctcaga
     5341 tcagatcgtg actcctcgag cggcggatgc tggcgaactg atctccgcca gcggaccgga
     5401 gatgagaccc cagcgaaccg ataacagagc gagagagctc cagttccgac tgattgcaca
     5461 gtcggtgatc tgggcgatgg gcactgccag ataggctggg aattatcaat cacttgaggt
     5521 gaaagtgcgg cgcacacaaa tccaagcttg atatcatcga tctcgaggct gcatccaacg
     5581 cgttgggagc tctccggatc aattcggctt caggtaccgt cgacgatgta ggtcacggtc
     5641 tcgaagccgc ggtgcgggtg ccagggcgtg cccttgggct ccccgggcgc gtactccacc
     5701 tcacccatct ggtccatcat gatgaacggg tcgaggtggc ggtagttgat cccggcgaac
     5761 gcgcggcgca ccgggaagcc ctcgccctcg aaaccgctgg gcgcggtggt cacggtgagc
     5821 acgggacgtg cgacggcgtc ggctgggtgc ggatacgcgg ggcagcgtca gcgggttctc
     5881 gacggtcacg gcgggcatgt cgacaagccg aattgatcca ctagaaggcc taattcggta
     5941 cactagttgg ccacgtaata agtgtgcgtt gaatttattc gcaaaaacat tgcatatttt
     6001 cggcaaagta aaattttgtt gcatacctta tcaaaaaata agtgctgcat actttttaga
     6061 gaaaccaaat aattttttat tgcatacccg tttttaataa aatacattgc ataccctctt
     6121 ttaataaaaa atattgcata ctttgacgaa acaaattttc gttgcatacc caataaaaga
     6181 ttattatatt gcatacccgt ttttaataaa atacattgca tagatct
//
