LOCUS       sequence-306364-        6389 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of pegRNA(s) and sgRNA(s) under control of Drosophila 
            U6:3 promoter. NS stands for ""No Scaffold"". Can be used to 
            generate transgenic flies with vermillion+ selection. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6389)
  AUTHORS   Bosch JA, Birchak G, Perrimon N
  TITLE     Precise genome engineering in Drosophila using prime editing.
  JOURNAL   Proc Natl Acad Sci U S A. 2021 Jan 5;118(1). pii: 2021996118. doi: 
            10.1073/pnas.2021996118.
  PUBMED    33443210
REFERENCE   2  (bases 1 to 6389)
  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."; date: "2021-01-5"; volume: "118(1). pii: 
            2021996118. doi"; pages: " 10.1073/pnas.2021996118"
COMMENT     Sequence Label: pCFD5-NS
FEATURES             Location/Qualifiers
     source          1..6389
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    31..52
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(169..186)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(340..928)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(420..439)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1099..1890,1891..1959))
                     /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     1722..1741
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1960..2064)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2091..2547
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2178..2197)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2388..2409
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2673..2695
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2687..2704
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2688..2704
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2714..2733
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2714..2732
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(join(2924..3041,3118..3211,3303..3909,3964..4097,
                     4158..4318,4392..4417))
                     /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    4732..4801
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     protein_bind    5043..5428
                     /label=gypsy insulator
                     /bound_moiety="Su(Hw), CP190, andMod(mdg4)67.2"
                     /note="chromatin insulator from Drosophila"
     promoter        5442..5862
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     primer_bind     complement(6288..6308)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6288..6306)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6327..6343)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6327..6343)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6340..6362)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6351..6367
                     /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(6375..6381,6382..6389,1..10,11..16))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
       61 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
      121 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
      181 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
      241 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
      301 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
      361 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
      421 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
      481 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
      541 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
      601 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
      661 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
      721 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
      781 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
      841 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
      901 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
      961 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     1021 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     1081 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     1141 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     1201 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     1261 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     1321 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     1381 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     1441 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     1501 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     1561 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     1621 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     1681 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     1741 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     1801 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     1861 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     1921 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     1981 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     2041 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgcgccctg
     2101 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     2161 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     2221 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     2281 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     2341 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     2401 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     2461 gccgatttcg gcctattggt taaaaaaatg agctgattta acaaaaattt aacgcgaatt
     2521 ttaacaaaat attaacgctt acaatttcca ttcgccattc aggctgcgca actgttggga
     2581 agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc
     2641 aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc
     2701 cagtgagcgc gcgtaatacg actcactata gggcgaattg ggtacaagct tggatttatt
     2761 ttgttatgtt atatgtatta tatgtcagac ataaagaaaa ggaacacatc aaatgtgata
     2821 acaaagacta aacaagtaat tttattacac caaaacgaca aaacagtagg cagaacaaac
     2881 aacgcatagc caaacattga cgaattggat accctgccga ttgtcagaca cttttgttga
     2941 tcagtttctt gcgaatggtc tcgtccagcg gtggaatcgc ctcgcgggga atcagaaaag
     3001 tggacagatt gaacagatcc agaaacacct tgtaccgatc actgaaacca aaaaaaaaca
     3061 aagggagaac agtttgagtt cattgatccc cgatataatc acatctgcga tgatcacctg
     3121 agagtggagc gcagatattg atatccagac gagccaccag tgcccaactg ttgggatcca
     3181 atcatgcgtt gcaccatgat cacgtgattg tctgcggcgg gaatagaaag tatttggtta
     3241 ggaaaaccag tcttaaacat aagatatatt tataaaagag tatcaaagaa tgcaatactt
     3301 acatctccac ttggttatta acgagtcgat gtccatgagc agggtgagca actggtgtgg
     3361 ttggctgaac ctgggttcat ccctatagaa ggtgatcatg atggctccct gaagggcacg
     3421 atggctaaac cggcgatccc cacgacgcac cagtgcatcg tgcactgccg gatcaaagat
     3481 ggagcgatac acctcgcgtc gcttctcaat gtccatgagg cggtagtttt tcgccttctc
     3541 cacgggctcc tccatggcgc tctgtacctg cgcctccagg aatcgatcga cgctctcctg
     3601 aaacttggcc cagaagttga agccactctc ctccagtccg ggcgtcctct ccagccatcg
     3661 ctgcactagc tccagtagcg agggatcttt ctccgagttg cgaatcgagt tccgcgcctc
     3721 ctcgtcgcta aagacatccg agtacttctg gttgtatctc acccgctgct ctgtcagaac
     3781 tcccagcttg ttctcgatca aacggaactg cagcgactga aaaccagatg cgggtgccag
     3841 gtacttgcgg aagtccatga agtctagcgg ggtcatggtc tccagaatgg gcacttggtc
     3901 caccaggagc tgtacaaagg aagttataaa cggattttgg taagagattc agaaagcact
     3961 cacttttaga atcagaacca ctcggttcag tcgcttgaca atctccagcg tcttggtttc
     4021 atcgatgacc tctgcatcca acatgtctcg tatggagtcg aactcaaaga tgatctgctt
     4081 gaaccaaagc tcgtaggctg tggcgaaggt acttaaatgc cattgagtgt tgtcatcaaa
     4141 gttgtaaacc tactcaccct ggtgcgtgat gatgaacaga tgctcatcgt gcacgggtcg
     4201 cttgtcctcc tcggacagca tacactgggc atccagcagt ttgtccagca tcagatactc
     4261 tccatagatt ttgcccactt ccgtggttaa tggcaccgcc gaatcatcgt gatcgtttct
     4321 gtatgggttt gaattgaatc gcagaactga agatcgattg gcattcctgg acagcacgtg
     4381 ctggtgctca cccgtttcct gcatagggac agctcatggt gcacagctca gatcagatcg
     4441 tgactcctcg agcggcggat gctggcgaac tgatctccgc cagcggaccg gagatgagac
     4501 cccagcgaac cgataacaga gcgagagagc tccagttccg actgattgca cagtcggtga
     4561 tctgggcgat gggcactgcc agataggctg ggaattatca atcacttgag gtgaaagtgc
     4621 ggcgcacaca aatccaagct tgatatcatc gatctcgagg ctgcatccaa cgcgttggga
     4681 gctctccgga tcaattcggc ttcaggtacc gtcgacgatg taggtcacgg tctcgaagcc
     4741 gcggtgcggg tgccagggcg tgcccttggg ctccccgggc gcgtactcca cctcacccat
     4801 ctggtccatc atgatgaacg ggtcgaggtg gcggtagttg atcccggcga acgcgcggcg
     4861 caccgggaag ccctcgccct cgaaaccgct gggcgcggtg gtcacggtga gcacgggacg
     4921 tgcgacggcg tcggctggtg cggatacgcg gggcagcgtc agcgggttct cgacggtcac
     4981 ggcgggcatg tcgacaagcc gaattgatcc actagaaggc ctaattcggt acactagttg
     5041 gccacgtaat aagtgtgcgt tgaatttatt cgcaaaaaca ttgcatattt tcggcaaagt
     5101 aaaattttgt tgcatacctt atcaaaaaat aagtgctgca tactttttag agaaaccaaa
     5161 taatttttta ttgcataccc gtttttaata aaatacattg cataccctct tttaataaaa
     5221 aatattgcat actttgacga aacaaatttt cgttgcatac ccaataaaag attattatat
     5281 tgcatacccg tttttaataa aatacattgc ataccctctt ttaataaaga atattgcata
     5341 cgttgacgaa acaaattttc gttgcatacc caataaaaga ttattatatt gcataccttt
     5401 tcttgccata ccatttagcc gatcaattga gatctgaatt cttttttgct cacctgtgat
     5461 tgctcctact caaatacaaa aacatcaaat tttctgtcaa taaagcatat ttatttatat
     5521 ttattttaca ggaaagaatt ccttttaaag tgtattttaa cctataatga aaaacgatta
     5581 aaaaaaatac ataaaataat tcgaaaattt ttgaatagcc caggttgata aaaattcatt
     5641 tcatacgttt tataacttat gcccctaagt attttttgac catagtgttt caattctaca
     5701 ttaattttac agagtagaat gaaacgccac ctactcagcc aagaggcgaa aaggttagct
     5761 cgccaagcag agagggcgcc agtgctcact actttttata attctcaact tctttttcca
     5821 gactcagttc gtatatatag acctattttc aatttaacgt cggggctttg agtgtgtgta
     5881 gacatcaagc atcggtggtt cagtggtaga atgctcgcct gccacgcggg cggcccgggt
     5941 tcgattcccg gccgatgcag ggtcttcgag aagacctttt tttgcctacc tggagcctga
     6001 gagttgttca ataaaataaa aatgtttcgt ttttttgctt tcgccagtat ttattatttt
     6061 tcatcaatat gtattcaatt tggtatgtat ttagtaattg taatatatag acaatggttt
     6121 tccgttgacg tacatacatc tgacgtgtgt ttatttagac ataatagtta tgttttcaca
     6181 tctttttaat gttcgcttaa tgcgtatgca ttctagacaa ttgtgctcgg caacagtata
     6241 tttgtggtgt gccaaccaac aacctgcagg agctccagct tttgttccct ttagtgaggg
     6301 ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     6361 ctcacaattc cacacaacat acgagccgg
//
