LOCUS       sequence-160505-        4579 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pCFD4d expresses white sgRNA-1 from a U6:3 promoter and the same 
            white sgRNA-1 from a U6:1 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4579)
  AUTHORS   Ge DT, Tipping C, Brodsky MH, Zamore PD
  TITLE     Rapid Screening for CRISPR-Directed Editing of the Drosophila Genome
            Using white Co-conversion.
  JOURNAL   G3 (Bethesda). 2016 Aug 26. pii: g3.116.032557. doi: 
            10.1534/g3.116.032557.
  PUBMED    27543296
REFERENCE   2  (bases 1 to 4579)
  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: "G3 
            (Bethesda). 2016 Aug 26. pii: g3.116.032557. doi: 
            10.1534/g3.116.032557."
COMMENT     Sequence Label: pCFD4d-U6-1:white1-U6-3:white1
FEATURES             Location/Qualifiers
     source          1..4579
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..803
                     /label=dU6-1 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-1 
                     snRNA (Port et al., 2014)"
     misc_RNA        824..899
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        900..1320
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_RNA        1341..1416
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1727..1747)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1727..1745)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1766..1782)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1766..1782)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1779..1801)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1790..1806
                     /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(1814..1820,1821..1838,1839..1844))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1859..1880
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1997..2014)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2168..2756)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2248..2267)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2927..3718,3719..3787))
                     /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     3550..3569
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3788..3892)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3918..4373)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4055..4076)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4267..4286
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4500..4522
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4514..4531
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4515..4531
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4538..4557
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4538..4556
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
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 tcgataccat tcctgctctt tgggttttag agctagaaat
      841 agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct
      901 tttttgctca cctgtgattg ctcctactca aatacaaaaa catcaaattt tctgtcaata
      961 aagcatattt atttatattt attttacagg aaagaattcc ttttaaagtg tattttaacc
     1021 tataatgaaa aacgattaaa aaaaatacat aaaataattc gaaaattttt gaatagccca
     1081 ggttgataaa aattcatttc atacgtttta taacttatgc ccctaagtat tttttgacca
     1141 tagtgtttca attctacatt aattttacag agtagaatga aacgccacct actcagccaa
     1201 gaggcgaaaa ggttagctcg ccaagcagag agggcgccag tgctcactac tttttataat
     1261 tctcaacttc tttttccaga ctcagttcgt atatatagac ctattttcaa tttaacgtcg
     1321 ataccattcc tgctctttgg gttttagagc tagaaatagc aagttaaaat aaggctagtc
     1381 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgcctacct ggagcctgag
     1441 agttgttcaa taaaataaaa atgtttcgtt tttttgcttt cgccagtatt tattattttt
     1501 catcaatatg tattcaattt ggtatgtatt tagtaattgt aatatataga caatggtttt
     1561 ccgttgacgt acatacatct gacgtgtgtt tatttagaca taatagttat gttttcacat
     1621 ctttttaatg ttcgcttaat gcgtatgcat tctagacaat tgtgctcggc aacagtatat
     1681 ttgtggtgtg ccaaccaaca acctgcagga gctccagctt ttgttccctt tagtgagggt
     1741 taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     1801 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     1861 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     1921 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     1981 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     2041 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     2101 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     2161 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     2221 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     2281 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     2341 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     2401 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     2461 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     2521 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     2581 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     2641 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     2701 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     2761 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     2821 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     2881 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     2941 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     3001 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     3061 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     3121 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     3181 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     3241 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     3301 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     3361 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     3421 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     3481 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     3541 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     3601 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     3661 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     3721 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     3781 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     3841 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     3901 cccgaaaagt gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     3961 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     4021 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     4081 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     4141 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     4201 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     4261 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     4321 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc
     4381 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg
     4441 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc
     4501 ccagtcacga cgttgtaaaa cgacggccag tgaattgtaa tacgactcac tatagggcga
     4561 attgggtaca agctgatct
//
