LOCUS       sequence-247913-        6370 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Drosophila expression of frame selector sgRNA 0 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_0
FEATURES             Location/Qualifiers
     source          1..6370
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    106..175
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     protein_bind    417..802
                     /label=gypsy insulator
                     /bound_moiety="Su(Hw), CP190, andMod(mdg4)67.2"
                     /note="chromatin insulator from Drosophila"
     promoter        816..1236
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_RNA        1257..1332
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1643..1663)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1643..1661)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1682..1698)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1682..1698)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1695..1717)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1706..1722
                     /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(1730..1736,1737..1754,1755..1760))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1775..1796
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1913..1930)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2084..2672)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2164..2183)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2843..3634,3635..3703))
                     /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     3466..3485
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3704..3808)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3835..4291
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3922..3941)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4132..4153
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4417..4439
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4432..4448
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4458..4477
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4458..4476
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(join(4668..4785,4862..4955,5047..5653,5708..5841,
                     5902..6062,6136..6161))
                     /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"
ORIGIN
        1 cacaaatcca agcttgatat catcgatctc gaggctgcat ccaacgcgtt gggagctctc
       61 cggatcaatt cggcttcagg taccgtcgac gatgtaggtc acggtctcga agccgcggtg
      121 cgggtgccag ggcgtgccct tgggctcccc gggcgcgtac tccacctcac ccatctggtc
      181 catcatgatg aacgggtcga ggtggcggta gttgatcccg gcgaacgcgc ggcgcaccgg
      241 gaagccctcg ccctcgaaac cgctgggcgc ggtggtcacg gtgagcacgg gacgtgcgac
      301 ggcgtcggct ggtgcggata cgcggggcag cgtcagcggg ttctcgacgg tcacggcggg
      361 catgtcgaca agccgaattg atccactaga aggcctaatt cggtacacta gttggccacg
      421 taataagtgt gcgttgaatt tattcgcaaa aacattgcat attttcggca aagtaaaatt
      481 ttgttgcata ccttatcaaa aaataagtgc tgcatacttt ttagagaaac caaataattt
      541 tttattgcat acccgttttt aataaaatac attgcatacc ctcttttaat aaaaaatatt
      601 gcatactttg acgaaacaaa ttttcgttgc atacccaata aaagattatt atattgcata
      661 cccgttttta ataaaataca ttgcataccc tcttttaata aagaatattg catacgttga
      721 cgaaacaaat tttcgttgca tacccaataa aagattatta tattgcatac cttttcttgc
      781 cataccattt agccgatcaa ttgagatctg aattcttttt tgctcacctg tgattgctcc
      841 tactcaaata caaaaacatc aaattttctg tcaataaagc atatttattt atatttattt
      901 tacaggaaag aattcctttt aaagtgtatt ttaacctata atgaaaaacg attaaaaaaa
      961 atacataaaa taattcgaaa atttttgaat agcccaggtt gataaaaatt catttcatac
     1021 gttttataac ttatgcccct aagtattttt tgaccatagt gtttcaattc tacattaatt
     1081 ttacagagta gaatgaaacg ccacctactc agccaagagg cgaaaaggtt agctcgccaa
     1141 gcagagaggg cgccagtgct cactactttt tataattctc aacttctttt tccagactca
     1201 gttcgtatat atagacctat tttcaattta acgtcggcca gtacccaaaa agcggggttt
     1261 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca
     1321 ccgagtcggt gcttttttgc ctacctggag cctgagagtt gttcaataaa ataaaaatgt
     1381 ttcgtttttt tgctttcgcc agtatttatt atttttcatc aatatgtatt caatttggta
     1441 tgtatttagt aattgtaata tatagacaat ggttttccgt tgacgtacat acatctgacg
     1501 tgtgtttatt tagacataat agttatgttt tcacatcttt ttaatgttcg cttaatgcgt
     1561 atgcattcta gacaattgtg ctcggcaaca gtatatttgt ggtgtgccaa ccaacaacct
     1621 gcaggagctc cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat
     1681 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     1741 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     1801 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     1861 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     1921 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     1981 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     2041 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     2101 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     2161 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     2221 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     2281 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     2341 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     2401 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     2461 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     2521 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     2581 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     2641 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     2701 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     2761 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     2821 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     2881 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     2941 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     3001 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     3061 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     3121 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     3181 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     3241 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     3301 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     3361 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     3421 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     3481 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     3541 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     3601 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     3661 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     3721 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     3781 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgcgc
     3841 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     3901 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     3961 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     4021 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     4081 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     4141 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     4201 ttttgccgat ttcggcctat tggttaaaaa aatgagctga tttaacaaaa atttaacgcg
     4261 aattttaaca aaatattaac gcttacaatt tccattcgcc attcaggctg cgcaactgtt
     4321 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg
     4381 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga
     4441 cggccagtga gcgcgcgtaa tacgactcac tatagggcga attgggtaca agcttggatt
     4501 tattttgtta tgttatatgt attatatgtc agacataaag aaaaggaaca catcaaatgt
     4561 gataacaaag actaaacaag taattttatt acaccaaaac gacaaaacag taggcagaac
     4621 aaacaacgca tagccaaaca ttgacgaatt ggataccctg ccgattgtca gacacttttg
     4681 ttgatcagtt tcttgcgaat ggtctcgtcc agcggtggaa tcgcctcgcg gggaatcaga
     4741 aaagtggaca gattgaacag atccagaaac accttgtacc gatcactgaa accaaaaaaa
     4801 aacaaaggga gaacagtttg agttcattga tccccgatat aatcacatct gcgatgatca
     4861 cctgagagtg gagcgcagat attgatatcc agacgagcca ccagtgccca actgttggga
     4921 tccaatcatg cgttgcacca tgatcacgtg attgtctgcg gcgggaatag aaagtatttg
     4981 gttaggaaaa ccagtcttaa acataagata tatttataaa agagtatcaa agaatgcaat
     5041 acttacatct ccacttggtt attaacgagt cgatgtccat gagcagggtg agcaactggt
     5101 gtggttggct gaacctgggt tcatccctat agaaggtgat catgatggct ccctgaaggg
     5161 cacgatggct aaaccggcga tccccacgac gcaccagtgc atcgtgcact gccggatcaa
     5221 agatggagcg atacacctcg cgtcgcttct caatgtccat gaggcggtag tttttcgcct
     5281 tctccacggg ctcctccatg gcgctctgta cctgcgcctc caggaatcga tcgacgctct
     5341 cctgaaactt ggcccagaag ttgaagccac tctcctccag tccgggcgtc ctctccagcc
     5401 atcgctgcac tagctccagt agcgagggat ctttctccga gttgcgaatc gagttccgcg
     5461 cctcctcgtc gctaaagaca tccgagtact tctggttgta tctcacccgc tgctctgtca
     5521 gaactcccag cttgttctcg atcaaacgga actgcagcga ctgaaaacca gatgcgggtg
     5581 ccaggtactt gcggaagtcc atgaagtcta gcggggtcat ggtctccaga atgggcactt
     5641 ggtccaccag gagctgtaca aaggaagtta taaacggatt ttggtaagag attcagaaag
     5701 cactcacttt tagaatcaga accactcggt tcagtcgctt gacaatctcc agcgtcttgg
     5761 tttcatcgat gacctctgca tccaacatgt ctcgtatgga gtcgaactca aagatgatct
     5821 gcttgaacca aagctcgtag gctgtggcga aggtacttaa atgccattga gtgttgtcat
     5881 caaagttgta aacctactca ccctggtgcg tgatgatgaa cagatgctca tcgtgcacgg
     5941 gtcgcttgtc ctcctcggac agcatacact gggcatccag cagtttgtcc agcatcagat
     6001 actctccata gattttgccc acttccgtgg ttaatggcac cgccgaatca tcgtgatcgt
     6061 ttctgtatgg gtttgaattg aatcgcagaa ctgaagatcg attggcattc ctggacagca
     6121 cgtgctggtg ctcacccgtt tcctgcatag ggacagctca tggtgcacag ctcagatcag
     6181 atcgtgactc ctcgagcggc ggatgctggc gaactgatct ccgccagcgg accggagatg
     6241 agaccccagc gaaccgataa cagagcgaga gagctccagt tccgactgat tgcacagtcg
     6301 gtgatctggg cgatgggcac tgccagatag gctgggaatt atcaatcact tgaggtgaaa
     6361 gtgcggcgca
//
