LOCUS       sequence-253121-        5466 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPaint universal donor plasmid for gene exon insertion. Encodes 
            T2A-Nluc-SV40 and 3xP3-RFP visible eye marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5466)
  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 5466)
  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-Nluc-3xP3-RFP
FEATURES             Location/Qualifiers
     source          1..5466
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             58..111
                     /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"
     CDS             118..633
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     intron          723..788
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             918..938
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    1210..1331
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    1332..1365
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      1591..1600
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1597..2274
                     /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(1727..1745)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(1777..1797)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     1995..2014
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2189..2212
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    2395..2516
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    2546..2579
                     /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(2598..2618)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2598..2616)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2637..2653)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2637..2653)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2650..2672)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2661..2677
                     /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(2685..2691,2692..2709,2710..2715))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2730..2751
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2868..2885)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3039..3627)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3119..3138)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3798..4589,4590..4658))
                     /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     4421..4440
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4659..4763)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4790..5246
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4877..4896)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5087..5108
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5372..5394
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5386..5403
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5387..5403
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5413..5432
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5413..5431
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ccgggccagt acccaaaaag cggggggtcc ggtggaagcg gaggtagcgc ggccgccgag
       61 ggccgcggca gcctgctgac ctgcggcgat gtggaggaga accccgggcc cgctagcatg
      121 gtcttcacac tcgaagattt cgttggggac tggcgacaga cagccggcta caacctggac
      181 caagtccttg aacagggagg tgtgtccagt ttgtttcaga atctcggggt gtccgtaact
      241 ccgatccaaa ggattgtcct gagcggtgaa aatgggctga agatcgacat ccatgtcatc
      301 atcccgtatg aaggtctgag cggcgaccaa atgggccaga tcgaaaaaat ttttaaggtg
      361 gtgtaccctg tggatgatca tcactttaag gtgatcctgc actatggcac actggtaatc
      421 gacggggtta cgccgaacat gatcgactat ttcggacggc cgtatgaagg catcgccgtg
      481 ttcgacggca aaaagatcac tgtaacaggg accctgtgga acggcaacaa aattatcgac
      541 gagcgcctga tcaaccccga cggctccctg ctgttccgag taaccatcaa cggagtgacc
      601 ggctggcggc tgtgcgaacg cattctggcg taaggtacct ctagaggatc tttgtgaagg
      661 aaccttactt ctgtggtgtg acataattgg acaaactacc tacagagatt taaagctcta
      721 aggtaaatat aaaattttta agtgtataat gtgttaaact actgattcta attgtttgtg
      781 tattttagat tccaacctat ggaactgatg aatgggagca gtggtggaat gcctttaatg
      841 aggaaaacct gttttgctca gaagaaatgc catctagtga tgatgaggct actgctgact
      901 ctcaacattc tactcctcca aaaaagaaga gaaaggtaga agaccccaag gactttcctt
      961 cagaattgct aagttttttg agtcatgctg tgtttagtaa tagaactctt gcttgctttg
     1021 ctatttacac cacaaaggaa aaagctgcac tgctatacaa gaaaattatg gaaaaatatt
     1081 tgatgtatag tgccttgact agagatcata atcagccata ccacatttgt agaggtttta
     1141 cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt
     1201 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     1261 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     1321 aatgtatctt aataacttcg tataatgtat gctatacgaa gttatcgtac gggatctaat
     1381 tcaattagag actaattcaa ttagagctaa ttcaattagg atccaagctt atcgatttcg
     1441 aaccctcgac cgccggagta taaatagagg cgcttcgtct acggagcgac aattcaattc
     1501 aaacaagcaa agtgaacacg tcgctaagcg aaagctaagc aaataaacaa gcgcagctga
     1561 acaagctaaa caatcggctc gaagccggtc gccaccatgg cctcctccga ggacgtcatc
     1621 aaggagttca tgcgcttcaa ggtgcgcatg gagggctccg tgaacggcca cgagttcgag
     1681 atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg
     1741 accaagggcg gccccctgcc cttcgcctgg gacatcctgt ccccccagtt ccagtacggc
     1801 tccaaggtgt acgtgaagca ccccgccgac atccccgact acaagaagct gtccttcccc
     1861 gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc
     1921 caggactcct ccctccagga cggctccttc atctacaagg tgaagttcat cggcgtgaac
     1981 ttcccctccg acggccccgt aatgcagaag aagactatgg gctgggaggc gtccaccgag
     2041 cgcctgtacc cccgcgacgg cgtgctgaag ggcgagatcc acaaggccct gaagctgaag
     2101 gacggcggcc actacctggt ggagttcaag tccatctaca tggccaagaa gcccgtgcag
     2161 ctgcccggct actactacgt ggactccaag ctggacatca cctcccacaa cgaggactac
     2221 accatcgtgg agcagtacga gcgcgccgag ggccgccacc acctgttcct gtaggggccg
     2281 cgactctaga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac
     2341 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg
     2401 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     2461 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaaccg
     2521 tctgtaagca gcgaaggccc atacgataac ttcgtataat gtatgctata cgaagttatg
     2581 agctccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca
     2641 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     2701 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     2761 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     2821 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     2881 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     2941 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     3001 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     3061 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     3121 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     3181 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     3241 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     3301 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     3361 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     3421 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     3481 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     3541 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     3601 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     3661 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     3721 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     3781 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     3841 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     3901 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     3961 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     4021 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     4081 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     4141 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     4201 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     4261 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     4321 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     4381 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     4441 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     4501 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     4561 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     4621 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     4681 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     4741 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgcgccctgt
     4801 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     4861 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     4921 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     4981 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     5041 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     5101 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     5161 ccgatttcgg cctattggtt aaaaaaatga gctgatttaa caaaaattta acgcgaattt
     5221 taacaaaata ttaacgctta caatttccat tcgccattca ggctgcgcaa ctgttgggaa
     5281 gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca
     5341 aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc
     5401 agtgagcgcg cgtaatacga ctcactatag ggcgaattgg gtacaagctt cacgacctga
     5461 ggcgcg
//
