LOCUS       sequence-248029-        5580 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPaint universal donor plasmid for gene exon insertion. Encodes 
            T2A-Gal4DBD-SV40 and 3xP3-RFP visible eye marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5580)
  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 5580)
  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-Gal4DBD-3xP3-RFP
FEATURES             Location/Qualifiers
     source          1..5580
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     39..61
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     53..70
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     54..70
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     80..99
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        80..98
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             191..244
                     /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             437..877
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 DNA binding domain
                     /translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
                     RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
                     DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     intron          970..1035
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             1165..1185
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    1457..1578
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    1579..1612
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      1838..1847
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1844..2521
                     /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(1974..1992)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(2024..2044)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     2242..2261
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2436..2459
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    2642..2763
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    2793..2826
                     /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(2845..2865)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2845..2863)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2884..2900)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2884..2900)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2897..2919)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2908..2924
                     /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(2932..2938,2939..2956,2957..2962))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2977..2998
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3115..3132)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3286..3874)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3366..3385)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4045..4836,4837..4905))
                     /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     4668..4687
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4906..5010)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5037..5493
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5124..5143)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5334..5355
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac
       61 gacggccagt gagcgcgcgt aatacgactc actatagggc gaattgggta caagcttcac
      121 gacctgaggc gcgccgggcc agtacccaaa aagcgggggg tccggtggaa gcggaggtag
      181 cgcggccgcc gagggccgcg gcagcctgct gacctgcggc gatgtggagg agaaccccgg
      241 gcccgctagc atgctggaga tccgcgccgc cttcctgcgc cagcgcaaca ccgccctgcg
      301 caccgaggtg gccgagctgg agcaggaggt gcagcgcctg gagaacgagg tgagccagta
      361 cgagacccgc tacggccccc tgggcggcgg caaggctagc ggaggaggtg gtggaggtgg
      421 aggtggaggt actagtatga agctgctgag tagtattgaa caagcgtgtg acatatgccg
      481 acttaagaag ctgaagtgca gcaaggaaaa gccgaagtgc gccaaatgtc ttaagaacaa
      541 ttgggagtgc cggtactcgc caaagacgaa gcgaagccct ttgacacgcg ctcatttgac
      601 tgaagtggag agtcgcctgg agcgccttga gcagttgttc ctcctcattt ttccccgtga
      661 agacctcgac atgatactga agatggatag tctccaagat attaaggccc tgctcactgg
      721 cctgtttgtt caggataacg tcaacaagga tgccgtgacg gaccgccttg cctcggtgga
      781 gacagatatg ccacttacct tgcggcagca tcgcatctcg gctaccagca gtagtgaaga
      841 aagttccaat aagggtcaac ggcaactgac ggtatcgtaa ggtacctcta gaggatcttt
      901 gtgaaggaac cttacttctg tggtgtgaca taattggaca aactacctac agagatttaa
      961 agctctaagg taaatataaa atttttaagt gtataatgtg ttaaactact gattctaatt
     1021 gtttgtgtat tttagattcc aacctatgga actgatgaat gggagcagtg gtggaatgcc
     1081 tttaatgagg aaaacctgtt ttgctcagaa gaaatgccat ctagtgatga tgaggctact
     1141 gctgactctc aacattctac tcctccaaaa aagaagagaa aggtagaaga ccccaaggac
     1201 tttccttcag aattgctaag ttttttgagt catgctgtgt ttagtaatag aactcttgct
     1261 tgctttgcta tttacaccac aaaggaaaaa gctgcactgc tatacaagaa aattatggaa
     1321 aaatatttga tgtatagtgc cttgactaga gatcataatc agccatacca catttgtaga
     1381 ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa
     1441 tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     1501 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     1561 actcatcaat gtatcttaat aacttcgtat aatgtatgct atacgaagtt atcgtacggg
     1621 atctaattca attagagact aattcaatta gagctaattc aattaggatc caagcttatc
     1681 gatttcgaac cctcgaccgc cggagtataa atagaggcgc ttcgtctacg gagcgacaat
     1741 tcaattcaaa caagcaaagt gaacacgtcg ctaagcgaaa gctaagcaaa taaacaagcg
     1801 cagctgaaca agctaaacaa tcggctcgaa gccggtcgcc accatggcct cctccgagga
     1861 cgtcatcaag gagttcatgc gcttcaaggt gcgcatggag ggctccgtga acggccacga
     1921 gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct
     1981 gaaggtgacc aagggcggcc ccctgccctt cgcctgggac atcctgtccc cccagttcca
     2041 gtacggctcc aaggtgtacg tgaagcaccc cgccgacatc cccgactaca agaagctgtc
     2101 cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac
     2161 cgtgacccag gactcctccc tccaggacgg ctccttcatc tacaaggtga agttcatcgg
     2221 cgtgaacttc ccctccgacg gccccgtaat gcagaagaag actatgggct gggaggcgtc
     2281 caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc gagatccaca aggccctgaa
     2341 gctgaaggac ggcggccact acctggtgga gttcaagtcc atctacatgg ccaagaagcc
     2401 cgtgcagctg cccggctact actacgtgga ctccaagctg gacatcacct cccacaacga
     2461 ggactacacc atcgtggagc agtacgagcg cgccgagggc cgccaccacc tgttcctgta
     2521 ggggccgcga ctctagatca taatcagcca taccacattt gtagaggttt tacttgcttt
     2581 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
     2641 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     2701 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     2761 ttaaccgtct gtaagcagcg aaggcccata cgataacttc gtataatgta tgctatacga
     2821 agttatgagc tccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc
     2881 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     2941 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     3001 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     3061 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     3121 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     3181 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     3241 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     3301 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     3361 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     3421 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     3481 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     3541 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     3601 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     3661 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     3721 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     3781 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     3841 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     3901 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     3961 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     4021 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     4081 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     4141 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     4201 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     4261 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     4321 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     4381 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     4441 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     4501 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     4561 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     4621 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     4681 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     4741 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     4801 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     4861 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     4921 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     4981 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgc
     5041 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     5101 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     5161 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     5221 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     5281 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     5341 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     5401 gattttgccg atttcggcct attggttaaa aaaatgagct gatttaacaa aaatttaacg
     5461 cgaattttaa caaaatatta acgcttacaa tttccattcg ccattcaggc tgcgcaactg
     5521 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg
//
