LOCUS       sequence-373034-        7631 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of nicking sgRNA and epegRNA under control of separate 
            Drosophila U6:2 and U6:3 promoters. 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 7631)
  TITLE     Optimized prime editing in Drosophila
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7631)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pU6:2-U6:3-tevopreq1
FEATURES             Location/Qualifiers
     source          1..7631
                     /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"
     promoter        501..921
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     terminator      complement(933..964)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     CDS             complement(981..1658)
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     RBS             1665..1676
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    1694..1712
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1719..1737
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     complement(2095..2115)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2095..2113)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2134..2150)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2134..2150)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2147..2169)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2158..2174
                     /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(2182..2188,2189..2206,2207..2212))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2227..2248
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2365..2382)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2536..3124)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2616..2635)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3295..4086,4087..4155))
                     /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     3918..3937
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4156..4260)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4287..4743
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4374..4393)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4584..4605
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4869..4891
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4883..4900
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4884..4900
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4910..4929
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4910..4928
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(join(5120..5237,5314..5407,5499..6105,6160..6293,
                     6354..6514,6588..6613))
                     /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    6928..6997
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     protein_bind    7239..7624
                     /label=gypsy insulator
                     /bound_moiety="Su(Hw), CP190, andMod(mdg4)67.2"
                     /note="chromatin insulator from Drosophila"
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 ttttttgctc acctgtgatt gctcctactc aaatacaaaa
      541 acatcaaatt ttctgtcaat aaagcatatt tatttatatt tattttacag gaaagaattc
      601 cttttaaagt gtattttaac ctataatgaa aaacgattaa aaaaaataca taaaataatt
      661 cgaaaatttt tgaatagccc aggttgataa aaattcattt catacgtttt ataacttatg
      721 cccctaagta ttttttgacc atagtgtttc aattctacat taattttaca gagtagaatg
      781 aaacgccacc tactcagcca agaggcgaaa aggttagctc gccaagcaga gagggcgcca
      841 gtgctcacta ctttttataa ttctcaactt ctttttccag actcagttcg tatatataga
      901 cctattttca atttaacgtc ggggtcttcc gtagtcaaaa gcctccggtc ggaggctttt
      961 gacttggctg aggagtgcca ttaagcaccg gtggagtgac gaccttcagc acgttcgtac
     1021 tgttcaacga tggtgtaatc ttcgttgtgg gaggtgatgt ccagtttgat gtcggttttg
     1081 taagcacccg gcagctgaac cggtttttta gccatgtagg tggttttaac ttcagcgtcg
     1141 tagtgaccac cgtctttcag tttcagacgc attttgattt cacctttcag agcaccatct
     1201 tccgggtaca tacgttcggt ggaagcttcc caacccatgg tttttttctg cataaccgga
     1261 ccgtcggacg ggaagttggt accacgcagt ttaactttgt agatgaactc accgtcttgc
     1321 agggaggagt cctgggtaac ggtaacaaca ccaccatctt cgaagttcat aacacgttcc
     1381 catttgaaac cttccgggaa ggacagtttc aggtagtccg ggatgtcagc cgggtgttta
     1441 acgtaagctt tggaaccgta ctggaactgc ggggacagga tgtcccaagc gaacggcagc
     1501 ggaccacctt tggtaacttt cagtttagcg gtctgggtac cttcgtacgg acgaccttca
     1561 ccttcacctt cgatttcgaa ctcgtgaccg ttaacggaac cttccatacg aactttgaaa
     1621 cgcatgaact ctttgataac atcttcgcta ctcgccatag atcatttctc ctctttcaga
     1681 tccgtgctca gtatctctat cactgatagg gatgtcaatc tctatcactg atagggagga
     1741 agacctcgcg gttctatcta gttacgcgtt aaaccaacta gaattttttt gcctacctgg
     1801 agcctgagag ttgttcaata aaataaaaat gtttcgtttt tttgctttcg ccagtattta
     1861 ttatttttca tcaatatgta ttcaatttgg tatgtattta gtaattgtaa tatatagaca
     1921 atggttttcc gttgacgtac atacatctga cgtgtgttta tttagacata atagttatgt
     1981 tttcacatct ttttaatgtt cgcttaatgc gtatgcattc tagacaattg tgctcggcaa
     2041 cagtatattt gtggtgtgcc aaccaacaac ctgcaggagc tccagctttt gttcccttta
     2101 gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     2161 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     2221 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     2281 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     2341 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     2401 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     2461 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     2521 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     2581 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     2641 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     2701 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     2761 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     2821 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     2881 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     2941 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     3001 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     3061 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     3121 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     3181 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     3241 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     3301 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     3361 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     3421 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     3481 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     3541 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     3601 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     3661 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     3721 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     3781 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     3841 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     3901 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     3961 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     4021 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     4081 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     4141 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     4201 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     4261 cacatttccc cgaaaagtgc cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg
     4321 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     4381 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     4441 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     4501 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     4561 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     4621 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     4681 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     4741 tttccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
     4801 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
     4861 agggttttcc cagtcacgac gttgtaaaac gacggccagt gagcgcgcgt aatacgactc
     4921 actatagggc gaattgggta caagcttgga tttattttgt tatgttatat gtattatatg
     4981 tcagacataa agaaaaggaa cacatcaaat gtgataacaa agactaaaca agtaatttta
     5041 ttacaccaaa acgacaaaac agtaggcaga acaaacaacg catagccaaa cattgacgaa
     5101 ttggataccc tgccgattgt cagacacttt tgttgatcag tttcttgcga atggtctcgt
     5161 ccagcggtgg aatcgcctcg cggggaatca gaaaagtgga cagattgaac agatccagaa
     5221 acaccttgta ccgatcactg aaaccaaaaa aaaacaaagg gagaacagtt tgagttcatt
     5281 gatccccgat ataatcacat ctgcgatgat cacctgagag tggagcgcag atattgatat
     5341 ccagacgagc caccagtgcc caactgttgg gatccaatca tgcgttgcac catgatcacg
     5401 tgattgtctg cggcgggaat agaaagtatt tggttaggaa aaccagtctt aaacataaga
     5461 tatatttata aaagagtatc aaagaatgca atacttacat ctccacttgg ttattaacga
     5521 gtcgatgtcc atgagcaggg tgagcaactg gtgtggttgg ctgaacctgg gttcatccct
     5581 atagaaggtg atcatgatgg ctccctgaag ggcacgatgg ctaaaccggc gatccccacg
     5641 acgcaccagt gcatcgtgca ctgccggatc aaagatggag cgatacacct cgcgtcgctt
     5701 ctcaatgtcc atgaggcggt agtttttcgc cttctccacg ggctcctcca tggcgctctg
     5761 tacctgcgcc tccaggaatc gatcgacgct ctcctgaaac ttggcccaga agttgaagcc
     5821 actctcctcc agtccgggcg tcctctccag ccatcgctgc actagctcca gtagcgaggg
     5881 atctttctcc gagttgcgaa tcgagttccg cgcctcctcg tcgctaaaga catccgagta
     5941 cttctggttg tatctcaccc gctgctctgt cagaactccc agcttgttct cgatcaaacg
     6001 gaactgcagc gactgaaaac cagatgcggg tgccaggtac ttgcggaagt ccatgaagtc
     6061 tagcggggtc atggtctcca gaatgggcac ttggtccacc aggagctgta caaaggaagt
     6121 tataaacgga ttttggtaag agattcagaa agcactcact tttagaatca gaaccactcg
     6181 gttcagtcgc ttgacaatct ccagcgtctt ggtttcatcg atgacctctg catccaacat
     6241 gtctcgtatg gagtcgaact caaagatgat ctgcttgaac caaagctcgt aggctgtggc
     6301 gaaggtactt aaatgccatt gagtgttgtc atcaaagttg taaacctact caccctggtg
     6361 cgtgatgatg aacagatgct catcgtgcac gggtcgcttg tcctcctcgg acagcataca
     6421 ctgggcatcc agcagtttgt ccagcatcag atactctcca tagattttgc ccacttccgt
     6481 ggttaatggc accgccgaat catcgtgatc gtttctgtat gggtttgaat tgaatcgcag
     6541 aactgaagat cgattggcat tcctggacag cacgtgctgg tgctcacccg tttcctgcat
     6601 agggacagct catggtgcac agctcagatc agatcgtgac tcctcgagcg gcggatgctg
     6661 gcgaactgat ctccgccagc ggaccggaga tgagacccca gcgaaccgat aacagagcga
     6721 gagagctcca gttccgactg attgcacagt cggtgatctg ggcgatgggc actgccagat
     6781 aggctgggaa ttatcaatca cttgaggtga aagtgcggcg cacacaaatc caagcttgat
     6841 atcatcgatc tcgaggctgc atccaacgcg ttgggagctc tccggatcaa ttcggcttca
     6901 ggtaccgtcg acgatgtagg tcacggtctc gaagccgcgg tgcgggtgcc agggcgtgcc
     6961 cttgggctcc ccgggcgcgt actccacctc acccatctgg tccatcatga tgaacgggtc
     7021 gaggtggcgg tagttgatcc cggcgaacgc gcggcgcacc gggaagccct cgccctcgaa
     7081 accgctgggc gcggtggtca cggtgagcac gggacgtgcg acggcgtcgg ctggtgcgga
     7141 tacgcggggc agcgtcagcg ggttctcgac ggtcacggcg ggcatgtcga caagccgaat
     7201 tgatccacta gaaggcctaa ttcggtacac tagttggcca cgtaataagt gtgcgttgaa
     7261 tttattcgca aaaacattgc atattttcgg caaagtaaaa ttttgttgca taccttatca
     7321 aaaaataagt gctgcatact ttttagagaa accaaataat tttttattgc atacccgttt
     7381 ttaataaaat acattgcata ccctctttta ataaaaaata ttgcatactt tgacgaaaca
     7441 aattttcgtt gcatacccaa taaaagatta ttatattgca tacccgtttt taataaaata
     7501 cattgcatac cctcttttaa taaagaatat tgcatacgtt gacgaaacaa attttcgttg
     7561 catacccaat aaaagattat tatattgcat accttttctt gccataccat ttagccgatc
     7621 aattgagatc t
//
