LOCUS       sequence-247916-        6370 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Drosophila expression of frame selector sgRNA 1 that targets 
            CRISPaint site for homology-independent knock-in.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6370)
  AUTHORS   Bosch JA, Colbeth R, Zirin J, Perrimon N
  TITLE     Gene Knock-Ins in Drosophila Using Homology-Independent Insertion of
            Universal Donor Plasmids.
  JOURNAL   Genetics. 2019 Nov 4. pii: genetics.119.302819. doi: 
            10.1534/genetics.119.302819.
  PUBMED    31685521
REFERENCE   2  (bases 1 to 6370)
  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: "Genetics. 
            2019 Nov 4. pii: genetics.119.302819. doi: 
            10.1534/genetics.119.302819."
COMMENT     Sequence Label: pCFD3-frame_selector_1
FEATURES             Location/Qualifiers
     source          1..6370
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        72..492
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_RNA        513..588
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(899..919)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(899..917)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(938..954)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(938..954)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(951..973)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    962..978
                     /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(986..992,993..1010,1011..1016))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1031..1052
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1169..1186)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1340..1928)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1420..1439)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2099..2890,2891..2959))
                     /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     2722..2741
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2960..3064)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3091..3547
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3178..3197)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3388..3409
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3673..3695
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3687..3704
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3688..3704
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3714..3733
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3714..3732
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(join(3924..4041,4118..4211,4303..4909,4964..5097,
                     5158..5318,5392..5417))
                     /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    5732..5801
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     protein_bind    join(6043..6370,1..58)
                     /label=gypsy insulator
                     /bound_moiety="Su(Hw), CP190, andMod(mdg4)67.2"
                     /note="chromatin insulator from Drosophila"
ORIGIN
        1 caataaaaga ttattatatt gcataccttt tcttgccata ccatttagcc gatcaattga
       61 gatctgaatt cttttttgct cacctgtgat tgctcctact caaatacaaa aacatcaaat
      121 tttctgtcaa taaagcatat ttatttatat ttattttaca ggaaagaatt ccttttaaag
      181 tgtattttaa cctataatga aaaacgatta aaaaaaatac ataaaataat tcgaaaattt
      241 ttgaatagcc caggttgata aaaattcatt tcatacgttt tataacttat gcccctaagt
      301 attttttgac catagtgttt caattctaca ttaattttac agagtagaat gaaacgccac
      361 ctactcagcc aagaggcgaa aaggttagct cgccaagcag agagggcgcc agtgctcact
      421 actttttata attctcaact tctttttcca gactcagttc gtatatatag acctattttc
      481 aatttaacgt cgggccagta cccaaaaagc gggttttaga gctagaaata gcaagttaaa
      541 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttgcctac
      601 ctggagcctg agagttgttc aataaaataa aaatgtttcg tttttttgct ttcgccagta
      661 tttattattt ttcatcaata tgtattcaat ttggtatgta tttagtaatt gtaatatata
      721 gacaatggtt ttccgttgac gtacatacat ctgacgtgtg tttatttaga cataatagtt
      781 atgttttcac atctttttaa tgttcgctta atgcgtatgc attctagaca attgtgctcg
      841 gcaacagtat atttgtggtg tgccaaccaa caacctgcag gagctccagc ttttgttccc
      901 tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa
      961 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     1021 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     1081 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     1141 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     1201 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     1261 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     1321 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     1381 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     1441 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     1501 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     1561 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     1621 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     1681 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     1741 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     1801 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     1861 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     1921 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     1981 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     2041 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     2101 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     2161 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     2221 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     2281 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     2341 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     2401 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     2461 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     2521 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     2581 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     2641 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     2701 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     2761 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     2821 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     2881 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     2941 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     3001 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     3061 cgcgcacatt tccccgaaaa gtgccacctg acgcgccctg tagcggcgca ttaagcgcgg
     3121 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     3181 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     3241 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     3301 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     3361 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     3421 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     3481 taaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     3541 acaatttcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
     3601 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa
     3661 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgagcgc gcgtaatacg
     3721 actcactata gggcgaattg ggtacaagct tggatttatt ttgttatgtt atatgtatta
     3781 tatgtcagac ataaagaaaa ggaacacatc aaatgtgata acaaagacta aacaagtaat
     3841 tttattacac caaaacgaca aaacagtagg cagaacaaac aacgcatagc caaacattga
     3901 cgaattggat accctgccga ttgtcagaca cttttgttga tcagtttctt gcgaatggtc
     3961 tcgtccagcg gtggaatcgc ctcgcgggga atcagaaaag tggacagatt gaacagatcc
     4021 agaaacacct tgtaccgatc actgaaacca aaaaaaaaca aagggagaac agtttgagtt
     4081 cattgatccc cgatataatc acatctgcga tgatcacctg agagtggagc gcagatattg
     4141 atatccagac gagccaccag tgcccaactg ttgggatcca atcatgcgtt gcaccatgat
     4201 cacgtgattg tctgcggcgg gaatagaaag tatttggtta ggaaaaccag tcttaaacat
     4261 aagatatatt tataaaagag tatcaaagaa tgcaatactt acatctccac ttggttatta
     4321 acgagtcgat gtccatgagc agggtgagca actggtgtgg ttggctgaac ctgggttcat
     4381 ccctatagaa ggtgatcatg atggctccct gaagggcacg atggctaaac cggcgatccc
     4441 cacgacgcac cagtgcatcg tgcactgccg gatcaaagat ggagcgatac acctcgcgtc
     4501 gcttctcaat gtccatgagg cggtagtttt tcgccttctc cacgggctcc tccatggcgc
     4561 tctgtacctg cgcctccagg aatcgatcga cgctctcctg aaacttggcc cagaagttga
     4621 agccactctc ctccagtccg ggcgtcctct ccagccatcg ctgcactagc tccagtagcg
     4681 agggatcttt ctccgagttg cgaatcgagt tccgcgcctc ctcgtcgcta aagacatccg
     4741 agtacttctg gttgtatctc acccgctgct ctgtcagaac tcccagcttg ttctcgatca
     4801 aacggaactg cagcgactga aaaccagatg cgggtgccag gtacttgcgg aagtccatga
     4861 agtctagcgg ggtcatggtc tccagaatgg gcacttggtc caccaggagc tgtacaaagg
     4921 aagttataaa cggattttgg taagagattc agaaagcact cacttttaga atcagaacca
     4981 ctcggttcag tcgcttgaca atctccagcg tcttggtttc atcgatgacc tctgcatcca
     5041 acatgtctcg tatggagtcg aactcaaaga tgatctgctt gaaccaaagc tcgtaggctg
     5101 tggcgaaggt acttaaatgc cattgagtgt tgtcatcaaa gttgtaaacc tactcaccct
     5161 ggtgcgtgat gatgaacaga tgctcatcgt gcacgggtcg cttgtcctcc tcggacagca
     5221 tacactgggc atccagcagt ttgtccagca tcagatactc tccatagatt ttgcccactt
     5281 ccgtggttaa tggcaccgcc gaatcatcgt gatcgtttct gtatgggttt gaattgaatc
     5341 gcagaactga agatcgattg gcattcctgg acagcacgtg ctggtgctca cccgtttcct
     5401 gcatagggac agctcatggt gcacagctca gatcagatcg tgactcctcg agcggcggat
     5461 gctggcgaac tgatctccgc cagcggaccg gagatgagac cccagcgaac cgataacaga
     5521 gcgagagagc tccagttccg actgattgca cagtcggtga tctgggcgat gggcactgcc
     5581 agataggctg ggaattatca atcacttgag gtgaaagtgc ggcgcacaca aatccaagct
     5641 tgatatcatc gatctcgagg ctgcatccaa cgcgttggga gctctccgga tcaattcggc
     5701 ttcaggtacc gtcgacgatg taggtcacgg tctcgaagcc gcggtgcggg tgccagggcg
     5761 tgcccttggg ctccccgggc gcgtactcca cctcacccat ctggtccatc atgatgaacg
     5821 ggtcgaggtg gcggtagttg atcccggcga acgcgcggcg caccgggaag ccctcgccct
     5881 cgaaaccgct gggcgcggtg gtcacggtga gcacgggacg tgcgacggcg tcggctggtg
     5941 cggatacgcg gggcagcgtc agcgggttct cgacggtcac ggcgggcatg tcgacaagcc
     6001 gaattgatcc actagaaggc ctaattcggt acactagttg gccacgtaat aagtgtgcgt
     6061 tgaatttatt cgcaaaaaca ttgcatattt tcggcaaagt aaaattttgt tgcatacctt
     6121 atcaaaaaat aagtgctgca tactttttag agaaaccaaa taatttttta ttgcataccc
     6181 gtttttaata aaatacattg cataccctct tttaataaaa aatattgcat actttgacga
     6241 aacaaatttt cgttgcatac ccaataaaag attattatat tgcatacccg tttttaataa
     6301 aatacattgc ataccctctt ttaataaaga atattgcata cgttgacgaa acaaattttc
     6361 gttgcatacc
//
