LOCUS       sequence-244414-        5663 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  CRISPaint universal donor plasmid for gene exon insertion. Encodes 
            T2A-sfGFP-SV40 and 3xP3-RFP visible eye marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5663)
  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 5663)
  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: pCRISPaint-T2A-sfGFP-3xP3-RFP
FEATURES             Location/Qualifiers
     source          1..5663
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     rep_origin      complement(6..461)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(143..164)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     355..374
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     588..610
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     602..619
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     603..619
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             737..790
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     intron          1603..1668
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             1798..1818
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2090..2211
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    2212..2245
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      2471..2480
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2477..3154
                     /codon_start=1
                     /product="rapidly maturing tetrameric variant of DsRed 
                     fluorescent protein (Bevis and Glick, 2002)"
                     /label=DsRed-Express
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAEGRHH
                     LFL"
     primer_bind     complement(2607..2625)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(2657..2677)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     2875..2894
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3069..3092
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    3275..3396
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    3426..3459
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        complement(3478..3496)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3517..3533)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3517..3533)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3530..3552)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3541..3557
                     /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(3565..3571,3572..3589,3590..3595))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3610..3631
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3748..3765)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3919..4507)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3999..4018)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4678..5469,5470..5538))
                     /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     5301..5320
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5539..5643)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
       61 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
      121 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
      181 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
      241 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
      301 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
      361 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
      421 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct
      481 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
      541 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
      601 ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tgggtacaag
      661 cttcacgacc tgaggcgcgc cgggccagta cccaaaaagc ggggggtccg gtggaagcgg
      721 aggtagcgcg gccgccgagg gccgcggcag cctgctgacc tgcggcgatg tggaggagaa
      781 ccccgggccc gctagcgtgt ccaagggcga ggagctgttc accggcgtgg tgcccatcct
      841 ggtggagctg gatggcgacg tgaacggcca caagttcagc gtgcgcggcg agggcgaggg
      901 cgacgccacc aacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
      961 gccctggccc accctggtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
     1021 cgatcacatg aagcagcacg atttcttcaa gagcgccatg cccgagggct acgtgcagga
     1081 gcgcaccatc agcttcaagg atgacggcac ctacaagacc cgcgccgagg tgaagttcga
     1141 gggcgatacc ctggtgaacc gcatcgagct gaagggcatc gatttcaagg aggatggcaa
     1201 catcctgggc cacaagctgg agtacaactt caacagccac aacgtgtaca tcaccgccga
     1261 taagcagaag aacggcatca aggccaactt caagatccgc cacaatgtgg aggatggctc
     1321 cgtgcagctg gccgatcact accagcagaa cacccccatc ggcgacggcc cagtgctgct
     1381 gcccgataac cactacctga gcacccagag cgtgctgtcc aaggacccca acgagaagcg
     1441 cgatcacatg gtgctgctgg agttcgtgac cgccgccggc atcaccctgg gcatggatga
     1501 gctgtacaag tagggtacct ctagaggatc tttgtgaagg aaccttactt ctgtggtgtg
     1561 acataattgg acaaactacc tacagagatt taaagctcta aggtaaatat aaaattttta
     1621 agtgtataat gtgttaaact actgattcta attgtttgtg tattttagat tccaacctat
     1681 ggaactgatg aatgggagca gtggtggaat gcctttaatg aggaaaacct gttttgctca
     1741 gaagaaatgc catctagtga tgatgaggct actgctgact ctcaacattc tactcctcca
     1801 aaaaagaaga gaaaggtaga agaccccaag gactttcctt cagaattgct aagttttttg
     1861 agtcatgctg tgtttagtaa tagaactctt gcttgctttg ctatttacac cacaaaggaa
     1921 aaagctgcac tgctatacaa gaaaattatg gaaaaatatt tgatgtatag tgccttgact
     1981 agagatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc
     2041 acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat
     2101 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     2161 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt aataacttcg
     2221 tataatgtat gctatacgaa gttatcgtac gggatctaat tcaattagag actaattcaa
     2281 ttagagctaa ttcaattagg atccaagctt atcgatttcg aaccctcgac cgccggagta
     2341 taaatagagg cgcttcgtct acggagcgac aattcaattc aaacaagcaa agtgaacacg
     2401 tcgctaagcg aaagctaagc aaataaacaa gcgcagctga acaagctaaa caatcggctc
     2461 gaagccggtc gccaccatgg cctcctccga ggacgtcatc aaggagttca tgcgcttcaa
     2521 ggtgcgcatg gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg
     2581 ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggcg gccccctgcc
     2641 cttcgcctgg gacatcctgt ccccccagtt ccagtacggc tccaaggtgt acgtgaagca
     2701 ccccgccgac atccccgact acaagaagct gtccttcccc gagggcttca agtgggagcg
     2761 cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctccagga
     2821 cggctccttc atctacaagg tgaagttcat cggcgtgaac ttcccctccg acggccccgt
     2881 aatgcagaag aagactatgg gctgggaggc gtccaccgag cgcctgtacc cccgcgacgg
     2941 cgtgctgaag ggcgagatcc acaaggccct gaagctgaag gacggcggcc actacctggt
     3001 ggagttcaag tccatctaca tggccaagaa gcccgtgcag ctgcccggct actactacgt
     3061 ggactccaag ctggacatca cctcccacaa cgaggactac accatcgtgg agcagtacga
     3121 gcgcgccgag ggccgccacc acctgttcct gtaggggccg cgactctaga tcataatcag
     3181 ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa
     3241 cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg
     3301 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     3361 tagttgtggt ttgtccaaac tcatcaatgt atcttaaccg tctgtaagca gcgaaggccc
     3421 atacgataac ttcgtataat gtatgctata cgaagttatg agctccagct tttgttccct
     3481 ttagtgaggg ttaatttcga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     3541 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     3601 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     3661 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     3721 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     3781 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     3841 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     3901 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     3961 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     4021 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     4081 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     4141 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     4201 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     4261 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     4321 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
     4381 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     4441 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     4501 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     4561 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     4621 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     4681 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     4741 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     4801 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     4861 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     4921 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     4981 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     5041 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     5101 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     5161 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     5221 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     5281 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     5341 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     5401 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     5461 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     5521 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     5581 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     5641 gcgcacattt ccccgaaaag tgc
//
