LOCUS       sequence-244393-        6370 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Drosophila expression of frame selector sgRNA 2 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_2
FEATURES             Location/Qualifiers
     source          1..6370
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(17..37)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(17..35)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(56..72)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(56..72)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(69..91)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    80..96
                     /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(104..110,111..128,129..134))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    149..170
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(287..304)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(458..1046)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(538..557)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1217..2008,2009..2077))
                     /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     1840..1859
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2078..2182)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2209..2665
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2296..2315)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2506..2527
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2791..2813
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2805..2822
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2806..2822
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2832..2851
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2832..2850
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(join(3042..3159,3236..3329,3421..4027,4082..4215,
                     4276..4436,4510..4535))
                     /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    4850..4919
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     protein_bind    5161..5546
                     /label=gypsy insulator
                     /bound_moiety="Su(Hw), CP190, andMod(mdg4)67.2"
                     /note="chromatin insulator from Drosophila"
     promoter        5560..5980
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_RNA        6001..6076
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 gctccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat
       61 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
      121 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
      181 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
      241 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
      301 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
      361 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
      421 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
      481 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
      541 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
      601 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
      661 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
      721 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
      781 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
      841 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
      901 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
      961 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     1021 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     1081 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     1141 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     1201 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     1261 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     1321 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     1381 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     1441 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     1501 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     1561 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     1621 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     1681 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     1741 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     1801 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     1861 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     1921 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     1981 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     2041 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     2101 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     2161 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gcgccctgta
     2221 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     2281 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     2341 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     2401 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     2461 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     2521 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     2581 cgatttcggc ctattggtta aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     2641 aacaaaatat taacgcttac aatttccatt cgccattcag gctgcgcaac tgttgggaag
     2701 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
     2761 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
     2821 gtgagcgcgc gtaatacgac tcactatagg gcgaattggg tacaagcttg gatttatttt
     2881 gttatgttat atgtattata tgtcagacat aaagaaaagg aacacatcaa atgtgataac
     2941 aaagactaaa caagtaattt tattacacca aaacgacaaa acagtaggca gaacaaacaa
     3001 cgcatagcca aacattgacg aattggatac cctgccgatt gtcagacact tttgttgatc
     3061 agtttcttgc gaatggtctc gtccagcggt ggaatcgcct cgcggggaat cagaaaagtg
     3121 gacagattga acagatccag aaacaccttg taccgatcac tgaaaccaaa aaaaaacaaa
     3181 gggagaacag tttgagttca ttgatccccg atataatcac atctgcgatg atcacctgag
     3241 agtggagcgc agatattgat atccagacga gccaccagtg cccaactgtt gggatccaat
     3301 catgcgttgc accatgatca cgtgattgtc tgcggcggga atagaaagta tttggttagg
     3361 aaaaccagtc ttaaacataa gatatattta taaaagagta tcaaagaatg caatacttac
     3421 atctccactt ggttattaac gagtcgatgt ccatgagcag ggtgagcaac tggtgtggtt
     3481 ggctgaacct gggttcatcc ctatagaagg tgatcatgat ggctccctga agggcacgat
     3541 ggctaaaccg gcgatcccca cgacgcacca gtgcatcgtg cactgccgga tcaaagatgg
     3601 agcgatacac ctcgcgtcgc ttctcaatgt ccatgaggcg gtagtttttc gccttctcca
     3661 cgggctcctc catggcgctc tgtacctgcg cctccaggaa tcgatcgacg ctctcctgaa
     3721 acttggccca gaagttgaag ccactctcct ccagtccggg cgtcctctcc agccatcgct
     3781 gcactagctc cagtagcgag ggatctttct ccgagttgcg aatcgagttc cgcgcctcct
     3841 cgtcgctaaa gacatccgag tacttctggt tgtatctcac ccgctgctct gtcagaactc
     3901 ccagcttgtt ctcgatcaaa cggaactgca gcgactgaaa accagatgcg ggtgccaggt
     3961 acttgcggaa gtccatgaag tctagcgggg tcatggtctc cagaatgggc acttggtcca
     4021 ccaggagctg tacaaaggaa gttataaacg gattttggta agagattcag aaagcactca
     4081 cttttagaat cagaaccact cggttcagtc gcttgacaat ctccagcgtc ttggtttcat
     4141 cgatgacctc tgcatccaac atgtctcgta tggagtcgaa ctcaaagatg atctgcttga
     4201 accaaagctc gtaggctgtg gcgaaggtac ttaaatgcca ttgagtgttg tcatcaaagt
     4261 tgtaaaccta ctcaccctgg tgcgtgatga tgaacagatg ctcatcgtgc acgggtcgct
     4321 tgtcctcctc ggacagcata cactgggcat ccagcagttt gtccagcatc agatactctc
     4381 catagatttt gcccacttcc gtggttaatg gcaccgccga atcatcgtga tcgtttctgt
     4441 atgggtttga attgaatcgc agaactgaag atcgattggc attcctggac agcacgtgct
     4501 ggtgctcacc cgtttcctgc atagggacag ctcatggtgc acagctcaga tcagatcgtg
     4561 actcctcgag cggcggatgc tggcgaactg atctccgcca gcggaccgga gatgagaccc
     4621 cagcgaaccg ataacagagc gagagagctc cagttccgac tgattgcaca gtcggtgatc
     4681 tgggcgatgg gcactgccag ataggctggg aattatcaat cacttgaggt gaaagtgcgg
     4741 cgcacacaaa tccaagcttg atatcatcga tctcgaggct gcatccaacg cgttgggagc
     4801 tctccggatc aattcggctt caggtaccgt cgacgatgta ggtcacggtc tcgaagccgc
     4861 ggtgcgggtg ccagggcgtg cccttgggct ccccgggcgc gtactccacc tcacccatct
     4921 ggtccatcat gatgaacggg tcgaggtggc ggtagttgat cccggcgaac gcgcggcgca
     4981 ccgggaagcc ctcgccctcg aaaccgctgg gcgcggtggt cacggtgagc acgggacgtg
     5041 cgacggcgtc ggctggtgcg gatacgcggg gcagcgtcag cgggttctcg acggtcacgg
     5101 cgggcatgtc gacaagccga attgatccac tagaaggcct aattcggtac actagttggc
     5161 cacgtaataa gtgtgcgttg aatttattcg caaaaacatt gcatattttc ggcaaagtaa
     5221 aattttgttg cataccttat caaaaaataa gtgctgcata ctttttagag aaaccaaata
     5281 attttttatt gcatacccgt ttttaataaa atacattgca taccctcttt taataaaaaa
     5341 tattgcatac tttgacgaaa caaattttcg ttgcataccc aataaaagat tattatattg
     5401 catacccgtt tttaataaaa tacattgcat accctctttt aataaagaat attgcatacg
     5461 ttgacgaaac aaattttcgt tgcataccca ataaaagatt attatattgc ataccttttc
     5521 ttgccatacc atttagccga tcaattgaga tctgaattct tttttgctca cctgtgattg
     5581 ctcctactca aatacaaaaa catcaaattt tctgtcaata aagcatattt atttatattt
     5641 attttacagg aaagaattcc ttttaaagtg tattttaacc tataatgaaa aacgattaaa
     5701 aaaaatacat aaaataattc gaaaattttt gaatagccca ggttgataaa aattcatttc
     5761 atacgtttta taacttatgc ccctaagtat tttttgacca tagtgtttca attctacatt
     5821 aattttacag agtagaatga aacgccacct actcagccaa gaggcgaaaa ggttagctcg
     5881 ccaagcagag agggcgccag tgctcactac tttttataat tctcaacttc tttttccaga
     5941 ctcagttcgt atatatagac ctattttcaa tttaacgtcg gggccagtac ccaaaaagcg
     6001 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
     6061 ggcaccgagt cggtgctttt ttgcctacct ggagcctgag agttgttcaa taaaataaaa
     6121 atgtttcgtt tttttgcttt cgccagtatt tattattttt catcaatatg tattcaattt
     6181 ggtatgtatt tagtaattgt aatatataga caatggtttt ccgttgacgt acatacatct
     6241 gacgtgtgtt tatttagaca taatagttat gttttcacat ctttttaatg ttcgcttaat
     6301 gcgtatgcat tctagacaat tgtgctcggc aacagtatat ttgtggtgtg ccaaccaaca
     6361 acctgcagga
//
