LOCUS       sequence-373031-        7137 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of single epegRNA under control of Drosophila U6:3 
            promoter. 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 7137)
  TITLE     Optimized prime editing in Drosophila
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7137)
  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            "Unpublished"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: pU6:3-tevopreq1
FEATURES             Location/Qualifiers
     source          1..7137
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..427
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     terminator      complement(439..470)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     CDS             complement(487..1164)
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     RBS             1171..1182
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    1200..1218
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1225..1243
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     complement(1601..1621)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1601..1619)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1640..1656)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1640..1656)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1653..1675)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1664..1680
                     /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(1688..1694,1695..1712,1713..1718))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1733..1754
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1871..1888)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2042..2630)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2122..2141)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2801..3592,3593..3661))
                     /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     3424..3443
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3662..3766)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3793..4249
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3880..3899)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4090..4111
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4375..4397
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4389..4406
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4390..4406
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4416..4435
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4416..4434
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(join(4626..4743,4820..4913,5005..5611,5666..5799,
                     5860..6020,6094..6119))
                     /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    6434..6503
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     protein_bind    6745..7130
                     /label=gypsy insulator
                     /bound_moiety="Su(Hw), CP190, andMod(mdg4)67.2"
                     /note="chromatin insulator from Drosophila"
ORIGIN
        1 gaattctttt ttgctcacct gtgattgctc ctactcaaat acaaaaacat caaattttct
       61 gtcaataaag catatttatt tatatttatt ttacaggaaa gaattccttt taaagtgtat
      121 tttaacctat aatgaaaaac gattaaaaaa aatacataaa ataattcgaa aatttttgaa
      181 tagcccaggt tgataaaaat tcatttcata cgttttataa cttatgcccc taagtatttt
      241 ttgaccatag tgtttcaatt ctacattaat tttacagagt agaatgaaac gccacctact
      301 cagccaagag gcgaaaaggt tagctcgcca agcagagagg gcgccagtgc tcactacttt
      361 ttataattct caacttcttt ttccagactc agttcgtata tatagaccta ttttcaattt
      421 aacgtcgggg tcttccgtag tcaaaagcct ccggtcggag gcttttgact tggctgagga
      481 gtgccattaa gcaccggtgg agtgacgacc ttcagcacgt tcgtactgtt caacgatggt
      541 gtaatcttcg ttgtgggagg tgatgtccag tttgatgtcg gttttgtaag cacccggcag
      601 ctgaaccggt tttttagcca tgtaggtggt tttaacttca gcgtcgtagt gaccaccgtc
      661 tttcagtttc agacgcattt tgatttcacc tttcagagca ccatcttccg ggtacatacg
      721 ttcggtggaa gcttcccaac ccatggtttt tttctgcata accggaccgt cggacgggaa
      781 gttggtacca cgcagtttaa ctttgtagat gaactcaccg tcttgcaggg aggagtcctg
      841 ggtaacggta acaacaccac catcttcgaa gttcataaca cgttcccatt tgaaaccttc
      901 cgggaaggac agtttcaggt agtccgggat gtcagccggg tgtttaacgt aagctttgga
      961 accgtactgg aactgcgggg acaggatgtc ccaagcgaac ggcagcggac cacctttggt
     1021 aactttcagt ttagcggtct gggtaccttc gtacggacga ccttcacctt caccttcgat
     1081 ttcgaactcg tgaccgttaa cggaaccttc catacgaact ttgaaacgca tgaactcttt
     1141 gataacatct tcgctactcg ccatagatca tttctcctct ttcagatccg tgctcagtat
     1201 ctctatcact gatagggatg tcaatctcta tcactgatag ggaggaagac ctcgcggttc
     1261 tatctagtta cgcgttaaac caactagaat ttttttgcct acctggagcc tgagagttgt
     1321 tcaataaaat aaaaatgttt cgtttttttg ctttcgccag tatttattat ttttcatcaa
     1381 tatgtattca atttggtatg tatttagtaa ttgtaatata tagacaatgg ttttccgttg
     1441 acgtacatac atctgacgtg tgtttattta gacataatag ttatgttttc acatcttttt
     1501 aatgttcgct taatgcgtat gcattctaga caattgtgct cggcaacagt atatttgtgg
     1561 tgtgccaacc aacaacctgc aggagctcca gcttttgttc cctttagtga gggttaattg
     1621 cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     1681 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     1741 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     1801 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     1861 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     1921 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     1981 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     2041 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     2101 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     2161 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     2221 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     2281 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     2341 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     2401 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     2461 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     2521 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     2581 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     2641 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     2701 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     2761 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     2821 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     2881 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     2941 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     3001 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     3061 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     3121 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     3181 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     3241 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     3301 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     3361 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     3421 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     3481 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     3541 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     3601 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     3661 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     3721 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     3781 aagtgccacc tgacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     3841 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     3901 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     3961 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     4021 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     4081 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     4141 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaaa tgagctgatt
     4201 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc cattcgccat
     4261 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc
     4321 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt
     4381 cacgacgttg taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat
     4441 tgggtacaag cttggattta ttttgttatg ttatatgtat tatatgtcag acataaagaa
     4501 aaggaacaca tcaaatgtga taacaaagac taaacaagta attttattac accaaaacga
     4561 caaaacagta ggcagaacaa acaacgcata gccaaacatt gacgaattgg ataccctgcc
     4621 gattgtcaga cacttttgtt gatcagtttc ttgcgaatgg tctcgtccag cggtggaatc
     4681 gcctcgcggg gaatcagaaa agtggacaga ttgaacagat ccagaaacac cttgtaccga
     4741 tcactgaaac caaaaaaaaa caaagggaga acagtttgag ttcattgatc cccgatataa
     4801 tcacatctgc gatgatcacc tgagagtgga gcgcagatat tgatatccag acgagccacc
     4861 agtgcccaac tgttgggatc caatcatgcg ttgcaccatg atcacgtgat tgtctgcggc
     4921 gggaatagaa agtatttggt taggaaaacc agtcttaaac ataagatata tttataaaag
     4981 agtatcaaag aatgcaatac ttacatctcc acttggttat taacgagtcg atgtccatga
     5041 gcagggtgag caactggtgt ggttggctga acctgggttc atccctatag aaggtgatca
     5101 tgatggctcc ctgaagggca cgatggctaa accggcgatc cccacgacgc accagtgcat
     5161 cgtgcactgc cggatcaaag atggagcgat acacctcgcg tcgcttctca atgtccatga
     5221 ggcggtagtt tttcgccttc tccacgggct cctccatggc gctctgtacc tgcgcctcca
     5281 ggaatcgatc gacgctctcc tgaaacttgg cccagaagtt gaagccactc tcctccagtc
     5341 cgggcgtcct ctccagccat cgctgcacta gctccagtag cgagggatct ttctccgagt
     5401 tgcgaatcga gttccgcgcc tcctcgtcgc taaagacatc cgagtacttc tggttgtatc
     5461 tcacccgctg ctctgtcaga actcccagct tgttctcgat caaacggaac tgcagcgact
     5521 gaaaaccaga tgcgggtgcc aggtacttgc ggaagtccat gaagtctagc ggggtcatgg
     5581 tctccagaat gggcacttgg tccaccagga gctgtacaaa ggaagttata aacggatttt
     5641 ggtaagagat tcagaaagca ctcactttta gaatcagaac cactcggttc agtcgcttga
     5701 caatctccag cgtcttggtt tcatcgatga cctctgcatc caacatgtct cgtatggagt
     5761 cgaactcaaa gatgatctgc ttgaaccaaa gctcgtaggc tgtggcgaag gtacttaaat
     5821 gccattgagt gttgtcatca aagttgtaaa cctactcacc ctggtgcgtg atgatgaaca
     5881 gatgctcatc gtgcacgggt cgcttgtcct cctcggacag catacactgg gcatccagca
     5941 gtttgtccag catcagatac tctccataga ttttgcccac ttccgtggtt aatggcaccg
     6001 ccgaatcatc gtgatcgttt ctgtatgggt ttgaattgaa tcgcagaact gaagatcgat
     6061 tggcattcct ggacagcacg tgctggtgct cacccgtttc ctgcataggg acagctcatg
     6121 gtgcacagct cagatcagat cgtgactcct cgagcggcgg atgctggcga actgatctcc
     6181 gccagcggac cggagatgag accccagcga accgataaca gagcgagaga gctccagttc
     6241 cgactgattg cacagtcggt gatctgggcg atgggcactg ccagataggc tgggaattat
     6301 caatcacttg aggtgaaagt gcggcgcaca caaatccaag cttgatatca tcgatctcga
     6361 ggctgcatcc aacgcgttgg gagctctccg gatcaattcg gcttcaggta ccgtcgacga
     6421 tgtaggtcac ggtctcgaag ccgcggtgcg ggtgccaggg cgtgcccttg ggctccccgg
     6481 gcgcgtactc cacctcaccc atctggtcca tcatgatgaa cgggtcgagg tggcggtagt
     6541 tgatcccggc gaacgcgcgg cgcaccggga agccctcgcc ctcgaaaccg ctgggcgcgg
     6601 tggtcacggt gagcacggga cgtgcgacgg cgtcggctgg tgcggatacg cggggcagcg
     6661 tcagcgggtt ctcgacggtc acggcgggca tgtcgacaag ccgaattgat ccactagaag
     6721 gcctaattcg gtacactagt tggccacgta ataagtgtgc gttgaattta ttcgcaaaaa
     6781 cattgcatat tttcggcaaa gtaaaatttt gttgcatacc ttatcaaaaa ataagtgctg
     6841 catacttttt agagaaacca aataattttt tattgcatac ccgtttttaa taaaatacat
     6901 tgcataccct cttttaataa aaaatattgc atactttgac gaaacaaatt ttcgttgcat
     6961 acccaataaa agattattat attgcatacc cgtttttaat aaaatacatt gcataccctc
     7021 ttttaataaa gaatattgca tacgttgacg aaacaaattt tcgttgcata cccaataaaa
     7081 gattattata ttgcatacct tttcttgcca taccatttag ccgatcaatt gagatct
//
