LOCUS       sequence-247978-        6222 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPaint universal donor plasmid for gene exon insertion. Encodes 
            T2A-FLPo-SV40 and 3xP3-RFP visible eye marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6222)
  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 6222)
  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-FLPo-3xP3-RFP
FEATURES             Location/Qualifiers
     source          1..6222
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     130..152
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     144..161
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     145..161
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     171..190
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        171..189
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             282..335
                     /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          1703..1768
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             1898..1918
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2190..2311
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    2312..2345
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      2571..2580
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2577..3254
                     /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(2707..2725)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(2757..2777)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     2975..2994
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3169..3192
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    3375..3496
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    3526..3559
                     /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(3578..3598)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3578..3596)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3617..3633)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3617..3633)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3630..3652)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3641..3657
                     /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(3665..3671,3672..3689,3690..3695))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3710..3731
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3848..3865)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4019..4607)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4099..4118)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4778..5569,5570..5638))
                     /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     5401..5420
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5639..5743)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5770..6199
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5857..5876)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6067..6088
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 atttccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt
       61 cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc
      121 cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgagcgcgcg taatacgact
      181 cactataggg cgaattgggt acaagcttca cgacctgagg cgcgccgggc cagtacccaa
      241 aaagcggggg gtccggtgga agcggaggta gcgcggccgc cgagggccgc ggcagcctgc
      301 tgacctgcgg cgatgtggag gagaaccccg ggcccgctag catgagccag ttcgacatcc
      361 tgtgcaagac cccccccaag gtgctggtgc ggcagttcgt ggagagattc gagaggccca
      421 gcggcgagaa gatcgccagc tgtgccgccg agctgaccta cctgtgctgg atgatcaccc
      481 acaacggcac cgccatcaag agggccacct tcatgagcta caacaccatc atcagcaaca
      541 gcctgagctt cgacatcgtg aacaagagcc tgcagttcaa gtacaagacc cagaaggcca
      601 ccatcctgga ggccagcctg aagaagctga tccccgcctg ggagttcacc atcatccctt
      661 acaacggcca gaagcaccag agcgacatca ccgacatcgt gtccagcctg cagctgcagt
      721 tcgagagcag cgaggaggcc gacaagggca acagccacag caagaagatg ctgaaggccc
      781 tgctgtccga gggcgagagc atctgggaga tcaccgagaa gatcctgaac agcttcgagt
      841 acaccagcag gttcaccaag accaagaccc tgtaccagtt cctgttcctg gccacattca
      901 tcaactgcgg caggttcagc gacatcaaga acgtggaccc caagagcttc aagctggtgc
      961 agaacaagta cctgggcgtg atcattcagt gcctggtgac cgagaccaag acaagcgtgt
     1021 ccaggcacat ctactttttc agcgccagag gcaggatcga ccccctggtg tacctggacg
     1081 agttcctgag gaacagcgag cccgtgctga agagagtgaa caggaccggc aacagcagca
     1141 gcaacaagca ggagtaccag ctgctgaagg acaacctggt gcgcagctac aacaaggccc
     1201 tgaagaagaa cgccccctac cccatcttcg ctatcaagaa cggccctaag agccacatcg
     1261 gcaggcacct gatgaccagc tttctgagca tgaagggcct gaccgagctg acaaacgtgg
     1321 tgggcaactg gagcgacaag agggcctccg ccgtggccag gaccacctac acccaccaga
     1381 tcaccgccat ccccgaccac tacttcgccc tggtgtccag gtactacgcc tacgacccca
     1441 tcagcaagga gatgatcgcc ctgaaggacg agaccaaccc catcgaggag tggcagcaca
     1501 tcgagcagct gaagggcagc gccgagggca gcatcagata ccccgcctgg aacggcatca
     1561 tcagccagga ggtgctggac tacctgagca gctacatcaa caggcggatc tgaggtacct
     1621 ctagaggatc tttgtgaagg aaccttactt ctgtggtgtg acataattgg acaaactacc
     1681 tacagagatt taaagctcta aggtaaatat aaaattttta agtgtataat gtgttaaact
     1741 actgattcta attgtttgtg tattttagat tccaacctat ggaactgatg aatgggagca
     1801 gtggtggaat gcctttaatg aggaaaacct gttttgctca gaagaaatgc catctagtga
     1861 tgatgaggct actgctgact ctcaacattc tactcctcca aaaaagaaga gaaaggtaga
     1921 agaccccaag gactttcctt cagaattgct aagttttttg agtcatgctg tgtttagtaa
     1981 tagaactctt gcttgctttg ctatttacac cacaaaggaa aaagctgcac tgctatacaa
     2041 gaaaattatg gaaaaatatt tgatgtatag tgccttgact agagatcata atcagccata
     2101 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga
     2161 aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca
     2221 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     2281 gtggtttgtc caaactcatc aatgtatctt aataacttcg tataatgtat gctatacgaa
     2341 gttatcgtac gggatctaat tcaattagag actaattcaa ttagagctaa ttcaattagg
     2401 atccaagctt atcgatttcg aaccctcgac cgccggagta taaatagagg cgcttcgtct
     2461 acggagcgac aattcaattc aaacaagcaa agtgaacacg tcgctaagcg aaagctaagc
     2521 aaataaacaa gcgcagctga acaagctaaa caatcggctc gaagccggtc gccaccatgg
     2581 cctcctccga ggacgtcatc aaggagttca tgcgcttcaa ggtgcgcatg gagggctccg
     2641 tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc
     2701 agaccgccaa gctgaaggtg accaagggcg gccccctgcc cttcgcctgg gacatcctgt
     2761 ccccccagtt ccagtacggc tccaaggtgt acgtgaagca ccccgccgac atccccgact
     2821 acaagaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg
     2881 gcggcgtggt gaccgtgacc caggactcct ccctccagga cggctccttc atctacaagg
     2941 tgaagttcat cggcgtgaac ttcccctccg acggccccgt aatgcagaag aagactatgg
     3001 gctgggaggc gtccaccgag cgcctgtacc cccgcgacgg cgtgctgaag ggcgagatcc
     3061 acaaggccct gaagctgaag gacggcggcc actacctggt ggagttcaag tccatctaca
     3121 tggccaagaa gcccgtgcag ctgcccggct actactacgt ggactccaag ctggacatca
     3181 cctcccacaa cgaggactac accatcgtgg agcagtacga gcgcgccgag ggccgccacc
     3241 acctgttcct gtaggggccg cgactctaga tcataatcag ccataccaca tttgtagagg
     3301 ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg
     3361 caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     3421 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     3481 tcatcaatgt atcttaaccg tctgtaagca gcgaaggccc atacgataac ttcgtataat
     3541 gtatgctata cgaagttatg agctccagct tttgttccct ttagtgaggg ttaattgcgc
     3601 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     3661 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     3721 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     3781 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     3841 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     3901 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     3961 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     4021 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     4081 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     4141 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4201 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     4261 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4321 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4381 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     4441 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     4501 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     4561 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     4621 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     4681 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     4741 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     4801 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     4861 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     4921 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     4981 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     5041 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     5101 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     5161 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     5221 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     5281 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     5341 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     5401 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     5461 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     5521 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     5581 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     5641 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     5701 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     5761 tgccacctga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     5821 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     5881 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     5941 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     6001 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     6061 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     6121 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaaatga gctgatttaa
     6181 caaaaattta acgcgaattt taacaaaata ttaacgctta ca
//
