LOCUS       sequence-247917-        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"
     protein_bind    107..176
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     protein_bind    418..803
                     /label=gypsy insulator
                     /bound_moiety="Su(Hw), CP190, andMod(mdg4)67.2"
                     /note="chromatin insulator from Drosophila"
     promoter        817..1237
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_RNA        1258..1333
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1644..1664)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1644..1662)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1683..1699)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1683..1699)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1696..1718)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1707..1723
                     /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(1731..1737,1738..1755,1756..1761))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1776..1797
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1914..1931)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2085..2673)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2165..2184)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2844..3635,3636..3704))
                     /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     3467..3486
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3705..3809)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3836..4292
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3923..3942)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4133..4154
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4418..4440
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4432..4449
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4433..4449
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4459..4478
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4459..4477
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(join(4669..4786,4863..4956,5048..5654,5709..5842,
                     5903..6063,6137..6162))
                     /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 acacaaatcc aagcttgata tcatcgatct cgaggctgca tccaacgcgt tgggagctct
       61 ccggatcaat tcggcttcag gtaccgtcga cgatgtaggt cacggtctcg aagccgcggt
      121 gcgggtgcca gggcgtgccc ttgggctccc cgggcgcgta ctccacctca cccatctggt
      181 ccatcatgat gaacgggtcg aggtggcggt agttgatccc ggcgaacgcg cggcgcaccg
      241 ggaagccctc gccctcgaaa ccgctgggcg cggtggtcac ggtgagcacg ggacgtgcga
      301 cggcgtcggc tggtgcggat acgcggggca gcgtcagcgg gttctcgacg gtcacggcgg
      361 gcatgtcgac aagccgaatt gatccactag aaggcctaat tcggtacact agttggccac
      421 gtaataagtg tgcgttgaat ttattcgcaa aaacattgca tattttcggc aaagtaaaat
      481 tttgttgcat accttatcaa aaaataagtg ctgcatactt tttagagaaa ccaaataatt
      541 ttttattgca tacccgtttt taataaaata cattgcatac cctcttttaa taaaaaatat
      601 tgcatacttt gacgaaacaa attttcgttg catacccaat aaaagattat tatattgcat
      661 acccgttttt aataaaatac attgcatacc ctcttttaat aaagaatatt gcatacgttg
      721 acgaaacaaa ttttcgttgc atacccaata aaagattatt atattgcata ccttttcttg
      781 ccataccatt tagccgatca attgagatct gaattctttt ttgctcacct gtgattgctc
      841 ctactcaaat acaaaaacat caaattttct gtcaataaag catatttatt tatatttatt
      901 ttacaggaaa gaattccttt taaagtgtat tttaacctat aatgaaaaac gattaaaaaa
      961 aatacataaa ataattcgaa aatttttgaa tagcccaggt tgataaaaat tcatttcata
     1021 cgttttataa cttatgcccc taagtatttt ttgaccatag tgtttcaatt ctacattaat
     1081 tttacagagt agaatgaaac gccacctact cagccaagag gcgaaaaggt tagctcgcca
     1141 agcagagagg gcgccagtgc tcactacttt ttataattct caacttcttt ttccagactc
     1201 agttcgtata tatagaccta ttttcaattt aacgtcgggg ccagtaccca aaaagcggtt
     1261 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
     1321 accgagtcgg tgcttttttg cctacctgga gcctgagagt tgttcaataa aataaaaatg
     1381 tttcgttttt ttgctttcgc cagtatttat tatttttcat caatatgtat tcaatttggt
     1441 atgtatttag taattgtaat atatagacaa tggttttccg ttgacgtaca tacatctgac
     1501 gtgtgtttat ttagacataa tagttatgtt ttcacatctt tttaatgttc gcttaatgcg
     1561 tatgcattct agacaattgt gctcggcaac agtatatttg tggtgtgcca accaacaacc
     1621 tgcaggagct ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca
     1681 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     1741 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     1801 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     1861 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     1921 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     1981 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     2041 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     2101 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     2161 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     2221 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     2281 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     2341 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     2401 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     2461 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     2521 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     2581 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     2641 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     2701 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     2761 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     2821 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     2881 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     2941 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     3001 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     3061 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     3121 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     3181 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     3241 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     3301 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     3361 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     3421 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     3481 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     3541 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     3601 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     3661 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     3721 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     3781 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgcg
     3841 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     3901 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     3961 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     4021 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     4081 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     4141 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     4201 attttgccga tttcggccta ttggttaaaa aaatgagctg atttaacaaa aatttaacgc
     4261 gaattttaac aaaatattaa cgcttacaat ttccattcgc cattcaggct gcgcaactgt
     4321 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
     4381 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
     4441 acggccagtg agcgcgcgta atacgactca ctatagggcg aattgggtac aagcttggat
     4501 ttattttgtt atgttatatg tattatatgt cagacataaa gaaaaggaac acatcaaatg
     4561 tgataacaaa gactaaacaa gtaattttat tacaccaaaa cgacaaaaca gtaggcagaa
     4621 caaacaacgc atagccaaac attgacgaat tggataccct gccgattgtc agacactttt
     4681 gttgatcagt ttcttgcgaa tggtctcgtc cagcggtgga atcgcctcgc ggggaatcag
     4741 aaaagtggac agattgaaca gatccagaaa caccttgtac cgatcactga aaccaaaaaa
     4801 aaacaaaggg agaacagttt gagttcattg atccccgata taatcacatc tgcgatgatc
     4861 acctgagagt ggagcgcaga tattgatatc cagacgagcc accagtgccc aactgttggg
     4921 atccaatcat gcgttgcacc atgatcacgt gattgtctgc ggcgggaata gaaagtattt
     4981 ggttaggaaa accagtctta aacataagat atatttataa aagagtatca aagaatgcaa
     5041 tacttacatc tccacttggt tattaacgag tcgatgtcca tgagcagggt gagcaactgg
     5101 tgtggttggc tgaacctggg ttcatcccta tagaaggtga tcatgatggc tccctgaagg
     5161 gcacgatggc taaaccggcg atccccacga cgcaccagtg catcgtgcac tgccggatca
     5221 aagatggagc gatacacctc gcgtcgcttc tcaatgtcca tgaggcggta gtttttcgcc
     5281 ttctccacgg gctcctccat ggcgctctgt acctgcgcct ccaggaatcg atcgacgctc
     5341 tcctgaaact tggcccagaa gttgaagcca ctctcctcca gtccgggcgt cctctccagc
     5401 catcgctgca ctagctccag tagcgaggga tctttctccg agttgcgaat cgagttccgc
     5461 gcctcctcgt cgctaaagac atccgagtac ttctggttgt atctcacccg ctgctctgtc
     5521 agaactccca gcttgttctc gatcaaacgg aactgcagcg actgaaaacc agatgcgggt
     5581 gccaggtact tgcggaagtc catgaagtct agcggggtca tggtctccag aatgggcact
     5641 tggtccacca ggagctgtac aaaggaagtt ataaacggat tttggtaaga gattcagaaa
     5701 gcactcactt ttagaatcag aaccactcgg ttcagtcgct tgacaatctc cagcgtcttg
     5761 gtttcatcga tgacctctgc atccaacatg tctcgtatgg agtcgaactc aaagatgatc
     5821 tgcttgaacc aaagctcgta ggctgtggcg aaggtactta aatgccattg agtgttgtca
     5881 tcaaagttgt aaacctactc accctggtgc gtgatgatga acagatgctc atcgtgcacg
     5941 ggtcgcttgt cctcctcgga cagcatacac tgggcatcca gcagtttgtc cagcatcaga
     6001 tactctccat agattttgcc cacttccgtg gttaatggca ccgccgaatc atcgtgatcg
     6061 tttctgtatg ggtttgaatt gaatcgcaga actgaagatc gattggcatt cctggacagc
     6121 acgtgctggt gctcacccgt ttcctgcata gggacagctc atggtgcaca gctcagatca
     6181 gatcgtgact cctcgagcgg cggatgctgg cgaactgatc tccgccagcg gaccggagat
     6241 gagaccccag cgaaccgata acagagcgag agagctccag ttccgactga ttgcacagtc
     6301 ggtgatctgg gcgatgggca ctgccagata ggctgggaat tatcaatcac ttgaggtgaa
     6361 agtgcggcgc
//
