LOCUS       sequence-306357-        6292 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of single pegRNA 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 6292)
  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 6292)
  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: pCFD3-NS
FEATURES             Location/Qualifiers
     source          1..6292
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    37..422
                     /label=gypsy insulator
                     /bound_moiety="Su(Hw), CP190, andMod(mdg4)67.2"
                     /note="chromatin insulator from Drosophila"
     promoter        436..856
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     primer_bind     complement(1185..1205)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1185..1203)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1224..1240)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1224..1240)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1237..1259)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1248..1264
                     /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(1272..1278,1279..1296,1297..1302))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1317..1338
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1455..1472)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1626..2214)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1706..1725)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2385..3176,3177..3245))
                     /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     3008..3027
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3246..3350)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3377..3833
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3464..3483)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3674..3695
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3959..3981
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3973..3990
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3974..3990
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4000..4019
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4000..4018
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(join(4210..4327,4404..4497,4589..5195,5250..5383,
                     5444..5604,5678..5703))
                     /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    6018..6087
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
ORIGIN
        1 atccactaga aggcctaatt cggtacacta gttggccacg taataagtgt gcgttgaatt
       61 tattcgcaaa aacattgcat attttcggca aagtaaaatt ttgttgcata ccttatcaaa
      121 aaataagtgc tgcatacttt ttagagaaac caaataattt tttattgcat acccgttttt
      181 aataaaatac attgcatacc ctcttttaat aaaaaatatt gcatactttg acgaaacaaa
      241 ttttcgttgc atacccaata aaagattatt atattgcata cccgttttta ataaaataca
      301 ttgcataccc tcttttaata aagaatattg catacgttga cgaaacaaat tttcgttgca
      361 tacccaataa aagattatta tattgcatac cttttcttgc cataccattt agccgatcaa
      421 ttgagatctg aattcttttt tgctcacctg tgattgctcc tactcaaata caaaaacatc
      481 aaattttctg tcaataaagc atatttattt atatttattt tacaggaaag aattcctttt
      541 aaagtgtatt ttaacctata atgaaaaacg attaaaaaaa atacataaaa taattcgaaa
      601 atttttgaat agcccaggtt gataaaaatt catttcatac gttttataac ttatgcccct
      661 aagtattttt tgaccatagt gtttcaattc tacattaatt ttacagagta gaatgaaacg
      721 ccacctactc agccaagagg cgaaaaggtt agctcgccaa gcagagaggg cgccagtgct
      781 cactactttt tataattctc aacttctttt tccagactca gttcgtatat atagacctat
      841 tttcaattta acgtcggggt cttcgagaag accttttttt gcctacctgg agcctgagag
      901 ttgttcaata aaataaaaat gtttcgtttt tttgctttcg ccagtattta ttatttttca
      961 tcaatatgta ttcaatttgg tatgtattta gtaattgtaa tatatagaca atggttttcc
     1021 gttgacgtac atacatctga cgtgtgttta tttagacata atagttatgt tttcacatct
     1081 ttttaatgtt cgcttaatgc gtatgcattc tagacaattg tgctcggcaa cagtatattt
     1141 gtggtgtgcc aaccaacaac ctgcaggagc tccagctttt gttcccttta gtgagggtta
     1201 attgcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     1261 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     1321 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     1381 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     1441 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     1501 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     1561 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     1621 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     1681 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     1741 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     1801 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     1861 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     1921 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     1981 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     2041 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     2101 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     2161 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     2221 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     2281 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     2341 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     2401 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     2461 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     2521 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     2581 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     2641 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     2701 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     2761 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     2821 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     2881 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     2941 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     3001 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     3061 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     3121 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     3181 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     3241 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     3301 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     3361 cgaaaagtgc cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     3421 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     3481 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     3541 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     3601 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     3661 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     3721 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaaatgagct
     3781 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttccattcg
     3841 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
     3901 cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc
     3961 cagtcacgac gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc
     4021 gaattgggta caagcttgga tttattttgt tatgttatat gtattatatg tcagacataa
     4081 agaaaaggaa cacatcaaat gtgataacaa agactaaaca agtaatttta ttacaccaaa
     4141 acgacaaaac agtaggcaga acaaacaacg catagccaaa cattgacgaa ttggataccc
     4201 tgccgattgt cagacacttt tgttgatcag tttcttgcga atggtctcgt ccagcggtgg
     4261 aatcgcctcg cggggaatca gaaaagtgga cagattgaac agatccagaa acaccttgta
     4321 ccgatcactg aaaccaaaaa aaaacaaagg gagaacagtt tgagttcatt gatccccgat
     4381 ataatcacat ctgcgatgat cacctgagag tggagcgcag atattgatat ccagacgagc
     4441 caccagtgcc caactgttgg gatccaatca tgcgttgcac catgatcacg tgattgtctg
     4501 cggcgggaat agaaagtatt tggttaggaa aaccagtctt aaacataaga tatatttata
     4561 aaagagtatc aaagaatgca atacttacat ctccacttgg ttattaacga gtcgatgtcc
     4621 atgagcaggg tgagcaactg gtgtggttgg ctgaacctgg gttcatccct atagaaggtg
     4681 atcatgatgg ctccctgaag ggcacgatgg ctaaaccggc gatccccacg acgcaccagt
     4741 gcatcgtgca ctgccggatc aaagatggag cgatacacct cgcgtcgctt ctcaatgtcc
     4801 atgaggcggt agtttttcgc cttctccacg ggctcctcca tggcgctctg tacctgcgcc
     4861 tccaggaatc gatcgacgct ctcctgaaac ttggcccaga agttgaagcc actctcctcc
     4921 agtccgggcg tcctctccag ccatcgctgc actagctcca gtagcgaggg atctttctcc
     4981 gagttgcgaa tcgagttccg cgcctcctcg tcgctaaaga catccgagta cttctggttg
     5041 tatctcaccc gctgctctgt cagaactccc agcttgttct cgatcaaacg gaactgcagc
     5101 gactgaaaac cagatgcggg tgccaggtac ttgcggaagt ccatgaagtc tagcggggtc
     5161 atggtctcca gaatgggcac ttggtccacc aggagctgta caaaggaagt tataaacgga
     5221 ttttggtaag agattcagaa agcactcact tttagaatca gaaccactcg gttcagtcgc
     5281 ttgacaatct ccagcgtctt ggtttcatcg atgacctctg catccaacat gtctcgtatg
     5341 gagtcgaact caaagatgat ctgcttgaac caaagctcgt aggctgtggc gaaggtactt
     5401 aaatgccatt gagtgttgtc atcaaagttg taaacctact caccctggtg cgtgatgatg
     5461 aacagatgct catcgtgcac gggtcgcttg tcctcctcgg acagcataca ctgggcatcc
     5521 agcagtttgt ccagcatcag atactctcca tagattttgc ccacttccgt ggttaatggc
     5581 accgccgaat catcgtgatc gtttctgtat gggtttgaat tgaatcgcag aactgaagat
     5641 cgattggcat tcctggacag cacgtgctgg tgctcacccg tttcctgcat agggacagct
     5701 catggtgcac agctcagatc agatcgtgac tcctcgagcg gcggatgctg gcgaactgat
     5761 ctccgccagc ggaccggaga tgagacccca gcgaaccgat aacagagcga gagagctcca
     5821 gttccgactg attgcacagt cggtgatctg ggcgatgggc actgccagat aggctgggaa
     5881 ttatcaatca cttgaggtga aagtgcggcg cacacaaatc caagcttgat atcatcgatc
     5941 tcgaggctgc atccaacgcg ttgggagctc tccggatcaa ttcggcttca ggtaccgtcg
     6001 acgatgtagg tcacggtctc gaagccgcgg tgcgggtgcc agggcgtgcc cttgggctcc
     6061 ccgggcgcgt actccacctc acccatctgg tccatcatga tgaacgggtc gaggtggcgg
     6121 tagttgatcc cggcgaacgc gcggcgcacc gggaagccct cgccctcgaa accgctgggc
     6181 gcggtggtca cggtgagcac gggacgtgcg acggcgtcgg ctggtgcgga tacgcggggc
     6241 agcgtcagcg ggttctcgac ggtcacggcg ggcatgtcga caagccgaat tg
//
