LOCUS       sequence-248024-        5607 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPaint universal donor plasmid for gene exon insertion. Encodes 
            sfGFP-SV40 and 3xP3-RFP visible eye marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5607)
  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 5607)
  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-sfGFP-3xP3-RFP
FEATURES             Location/Qualifiers
     source          1..5607
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     85..107
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     99..116
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     100..116
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     126..145
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        126..144
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     intron          1043..1108
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             1238..1258
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    1530..1651
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    1652..1685
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      1911..1920
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1917..2594
                     /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(2047..2065)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(2097..2117)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     2315..2334
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2509..2532
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    2715..2836
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    2866..2899
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(2918..2938)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2918..2936)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2957..2973)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2957..2973)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2970..2992)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2981..2997
                     /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(3005..3011,3012..3029,3030..3035))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3050..3071
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3188..3205)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3359..3947)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3439..3458)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4118..4909,4910..4978))
                     /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     4741..4760
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4979..5083)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5110..5566
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5197..5216)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5407..5428
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat
       61 taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgagc
      121 gcgcgtaata cgactcacta tagggcgaat tgggtacaag cttcacgacc tgaggcgcgc
      181 cgggccagta cccaaaaagc ggggggtccg gtggaagcgg aggtagcgcg gccgccgtgt
      241 ccaagggcga ggagctgttc accggcgtgg tgcccatcct ggtggagctg gatggcgacg
      301 tgaacggcca caagttcagc gtgcgcggcg agggcgaggg cgacgccacc aacggcaagc
      361 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctggtga
      421 ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgatcacatg aagcagcacg
      481 atttcttcaa gagcgccatg cccgagggct acgtgcagga gcgcaccatc agcttcaagg
      541 atgacggcac ctacaagacc cgcgccgagg tgaagttcga gggcgatacc ctggtgaacc
      601 gcatcgagct gaagggcatc gatttcaagg aggatggcaa catcctgggc cacaagctgg
      661 agtacaactt caacagccac aacgtgtaca tcaccgccga taagcagaag aacggcatca
      721 aggccaactt caagatccgc cacaatgtgg aggatggctc cgtgcagctg gccgatcact
      781 accagcagaa cacccccatc ggcgacggcc cagtgctgct gcccgataac cactacctga
      841 gcacccagag cgtgctgtcc aaggacccca acgagaagcg cgatcacatg gtgctgctgg
      901 agttcgtgac cgccgccggc atcaccctgg gcatggatga gctgtacaag tagggtacct
      961 ctagaggatc tttgtgaagg aaccttactt ctgtggtgtg acataattgg acaaactacc
     1021 tacagagatt taaagctcta aggtaaatat aaaattttta agtgtataat gtgttaaact
     1081 actgattcta attgtttgtg tattttagat tccaacctat ggaactgatg aatgggagca
     1141 gtggtggaat gcctttaatg aggaaaacct gttttgctca gaagaaatgc catctagtga
     1201 tgatgaggct actgctgact ctcaacattc tactcctcca aaaaagaaga gaaaggtaga
     1261 agaccccaag gactttcctt cagaattgct aagttttttg agtcatgctg tgtttagtaa
     1321 tagaactctt gcttgctttg ctatttacac cacaaaggaa aaagctgcac tgctatacaa
     1381 gaaaattatg gaaaaatatt tgatgtatag tgccttgact agagatcata atcagccata
     1441 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga
     1501 aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca
     1561 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     1621 gtggtttgtc caaactcatc aatgtatctt aataacttcg tataatgtat gctatacgaa
     1681 gttatcgtac gggatctaat tcaattagag actaattcaa ttagagctaa ttcaattagg
     1741 atccaagctt atcgatttcg aaccctcgac cgccggagta taaatagagg cgcttcgtct
     1801 acggagcgac aattcaattc aaacaagcaa agtgaacacg tcgctaagcg aaagctaagc
     1861 aaataaacaa gcgcagctga acaagctaaa caatcggctc gaagccggtc gccaccatgg
     1921 cctcctccga ggacgtcatc aaggagttca tgcgcttcaa ggtgcgcatg gagggctccg
     1981 tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc
     2041 agaccgccaa gctgaaggtg accaagggcg gccccctgcc cttcgcctgg gacatcctgt
     2101 ccccccagtt ccagtacggc tccaaggtgt acgtgaagca ccccgccgac atccccgact
     2161 acaagaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg
     2221 gcggcgtggt gaccgtgacc caggactcct ccctccagga cggctccttc atctacaagg
     2281 tgaagttcat cggcgtgaac ttcccctccg acggccccgt aatgcagaag aagactatgg
     2341 gctgggaggc gtccaccgag cgcctgtacc cccgcgacgg cgtgctgaag ggcgagatcc
     2401 acaaggccct gaagctgaag gacggcggcc actacctggt ggagttcaag tccatctaca
     2461 tggccaagaa gcccgtgcag ctgcccggct actactacgt ggactccaag ctggacatca
     2521 cctcccacaa cgaggactac accatcgtgg agcagtacga gcgcgccgag ggccgccacc
     2581 acctgttcct gtaggggccg cgactctaga tcataatcag ccataccaca tttgtagagg
     2641 ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg
     2701 caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     2761 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     2821 tcatcaatgt atcttaaccg tctgtaagca gcgaaggccc atacgataac ttcgtataat
     2881 gtatgctata cgaagttatg agctccagct tttgttccct ttagtgaggg ttaattgcgc
     2941 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     3001 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     3061 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     3121 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     3181 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     3241 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     3301 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     3361 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     3421 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     3481 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     3541 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     3601 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     3661 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     3721 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     3781 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     3841 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     3901 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     3961 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     4021 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     4081 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     4141 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     4201 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     4261 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     4321 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     4381 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     4441 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     4501 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     4561 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     4621 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     4681 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     4741 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     4801 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     4861 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     4921 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     4981 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     5041 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     5101 tgccacctga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     5161 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     5221 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     5281 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     5341 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     5401 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     5461 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaaatga gctgatttaa
     5521 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttccat tcgccattca
     5581 ggctgcgcaa ctgttgggaa gggcgat
//
