LOCUS       sequence-253122-        7596 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPaint universal donor plasmid for gene exon insertion. Encodes 
            T2A-Gal4-SV40 and 3xP3-RFP visible eye marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7596)
  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 7596)
  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-Gal4-3xP3-RFP
FEATURES             Location/Qualifiers
     source          1..7596
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     77..99
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     91..108
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     92..108
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     118..137
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        118..136
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             229..282
                     /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             289..2934
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4"
                     /product="GAL4 transcriptional activator"
                     /label=GAL4
                     /translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
                     RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
                     DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVSIDSAAHHDNSTIPLDF
                     MPRDALHGFDWSEEDDMSDGLPFLKTDPNNNGFFGDGSLLCILRSIGFKPENYTNSNVN
                     RLPTMITDRYTLASRSTTSRLLQSYLNNFHPYCPIVHSPTLMMLYNNQIEIASKDQWQI
                     LFNCILAIGAWCIEGESTDIDVFYYQNAKSHLTSKVFESGSIILVTALHLLSRYTQWRQ
                     KTNTSYNFHSFSIRMAISLGLNRDLPSSFSDSSILEQRRRIWWSVYSWEIQLSLLYGRS
                     IQLSQNTISFPSSVDDVQRTTTGPTIYHGIIETARLLQVFTKIYELDKTVTAEKSPICA
                     KKCLMICNEIEEVSRQAPKFLQMDISTTALTNLLKEHPWLSFTRFELKWKQLSLIIYVL
                     RDFFTNFTQKKSQLEQDQNDHQSYEVKRCSIMLSDAAQRTVMSVSSYMDNHNVTPYFAW
                     NCSYYLFNAVLVPIKTLLSNSKSNAENNETAQLLQQINTVLMLLKKLATFKIQTCEKYI
                     QVLEEVCAPFLLSQCAIPLPHISYNNSNGSAIKNIVGSATIAQYPTLPEENVNNISVKY
                     VSPGSVGPSPVPLKSGASFSDLVKLLSNRPPSRNSPVTIPRSTPSHRSVTPFLGQQQQL
                     QSLVPLTPSALFGGANFNQSGNIADSSLSFTFTNSSNGPNLITTQTNSQALSQPIASSN
                     VHDNFMNNEITASKIDDGNNSKPLSPGWTDQTAYNAFGITTGMFNTTTMDDVYNYLFDD
                     EDTPPNPKKE"
     intron          3024..3089
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             3219..3239
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3511..3632
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    3633..3666
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      3892..3901
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3898..4575
                     /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(4028..4046)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(4078..4098)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     4296..4315
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4490..4513
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    4696..4817
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    4847..4880
                     /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(4899..4919)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4899..4917)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4938..4954)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4938..4954)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4951..4973)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4962..4978
                     /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(4986..4992,4993..5010,5011..5016))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5031..5052
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5169..5186)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5340..5928)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5420..5439)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6099..6890,6891..6959))
                     /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     6722..6741
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6960..7064)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      7091..7547
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7178..7197)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7388..7409
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg
       61 gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga gcgcgcgtaa
      121 tacgactcac tatagggcga attgggtaca agcttcacga cctgaggcgc gccgggccag
      181 tacccaaaaa gcggggggtc cggtggaagc ggaggtagcg cggccgccga gggccgcggc
      241 agcctgctga cctgcggcga tgtggaggag aaccccgggc ccgctagcat gaagctactg
      301 tcttctatcg aacaagcatg cgatatttgc cgacttaaaa agctcaagtg ctccaaagaa
      361 aaaccgaagt gcgccaagtg tctgaagaac aactgggagt gtcgctactc tcccaaaacc
      421 aaaaggtctc cgctgactag ggcacatctg acagaagtgg aatcaaggct agaaagactg
      481 gaacagctat ttctactgat ttttcctcga gaagaccttg acatgatttt gaaaatggat
      541 tctttacagg atataaaagc attgttaaca ggattatttg tacaagataa tgtgaataaa
      601 gatgccgtca cagatagatt ggcttcagtg gagactgata tgcctctaac attgagacag
      661 catagaataa gtgcgacatc atcatcggaa gagagtagta acaaaggtca aagacagttg
      721 actgtatcga ttgactcggc agctcatcat gataactcca caattccgtt ggattttatg
      781 cccagggatg ctcttcatgg atttgattgg tctgaagagg atgacatgtc ggatggcttg
      841 cccttcctga aaacggaccc caacaataat gggttctttg gcgacggttc tctcttatgt
      901 attcttcgat ctattggctt taaaccggaa aattacacga actctaacgt taacaggctc
      961 ccgaccatga ttacggatag atacacgttg gcttctagat ccacaacatc ccgtttactt
     1021 caaagttatc tcaataattt tcacccctac tgccctatcg tgcactcacc gacgctaatg
     1081 atgttgtata ataaccagat tgaaatcgcg tcgaaggatc aatggcaaat cctttttaac
     1141 tgcatattag ccattggagc ctggtgtata gagggggaat ctactgatat agatgttttt
     1201 tactatcaaa atgctaaatc tcatttgacg agcaaggtct tcgagtcagg ttccataatt
     1261 ttggtgacag ccctacatct tctgtcgcga tatacacagt ggaggcagaa aacaaatact
     1321 agctataatt ttcacagctt ttccataaga atggccatat cattgggctt gaatagggac
     1381 ctcccctcgt ccttcagtga tagcagcatt ctggaacaaa gacgccgaat ttggtggtct
     1441 gtctactctt gggagatcca attgtccctg ctttatggtc gatccatcca gctttctcag
     1501 aatacaatct ccttcccttc ttctgtcgac gatgtgcagc gtaccacaac aggtcccacc
     1561 atatatcatg gcatcattga aacagcaagg ctcttacaag ttttcacaaa aatctatgaa
     1621 ctagacaaaa cagtaactgc agaaaaaagt cctatatgtg caaaaaaatg cttgatgatt
     1681 tgtaatgaga ttgaggaggt ttcgagacag gcaccaaagt ttttacaaat ggatatttcc
     1741 accaccgctc taaccaattt gttgaaggaa cacccttggc tatcctttac aagattcgaa
     1801 ctgaagtgga aacagttgtc tcttatcatt tatgtattaa gagatttttt cactaatttt
     1861 acccagaaaa agtcacaact agaacaggat caaaatgatc atcaaagtta tgaagttaaa
     1921 cgatgctcca tcatgttaag cgatgcagca caaagaactg ttatgtctgt aagtagctat
     1981 atggacaatc ataatgtcac cccatatttt gcctggaatt gttcttatta cttgttcaat
     2041 gcagtcctag tacccataaa gactctactc tcaaactcaa aatcgaatgc tgagaataac
     2101 gagaccgcac aattattaca acaaattaac actgttctga tgctattaaa aaaactggcc
     2161 acttttaaaa tccagacttg tgaaaaatac attcaagtac tggaagaggt atgtgcgccg
     2221 tttctgttat cacagtgtgc aatcccatta ccgcatatca gttataacaa tagtaatggt
     2281 agcgccatta aaaatattgt cggttctgca actatcgccc aataccctac tcttccggag
     2341 gaaaatgtca acaatatcag tgttaaatat gtttctcctg gctcagtagg gccttcacct
     2401 gtgccattga aatcaggagc aagtttcagt gatctagtca agctgttatc taaccgtcca
     2461 ccctctcgta actctccagt gacaatacca agaagcacac cttcgcatcg ctcagtcacg
     2521 ccttttctag ggcaacagca acagctgcaa tcattagtgc cactgacccc gtctgctttg
     2581 tttggtggcg ccaattttaa tcaaagtggg aatattgctg atagctcatt gtccttcact
     2641 ttcactaaca gtagcaacgg tccgaacctc ataacaactc aaacaaattc tcaagcgctt
     2701 tcacaaccaa ttgcctcctc taacgttcat gataacttca tgaataatga aatcacggct
     2761 agtaaaattg atgatggtaa taattcaaaa ccactgtcac ctggttggac ggaccaaact
     2821 gcgtataacg cgtttggaat cactacaggg atgtttaata ccactacaat ggatgatgta
     2881 tataactatc tattcgatga tgaagatacc ccaccaaacc caaaaaaaga gtaaggtacc
     2941 tctagaggat ctttgtgaag gaaccttact tctgtggtgt gacataattg gacaaactac
     3001 ctacagagat ttaaagctct aaggtaaata taaaattttt aagtgtataa tgtgttaaac
     3061 tactgattct aattgtttgt gtattttaga ttccaaccta tggaactgat gaatgggagc
     3121 agtggtggaa tgcctttaat gaggaaaacc tgttttgctc agaagaaatg ccatctagtg
     3181 atgatgaggc tactgctgac tctcaacatt ctactcctcc aaaaaagaag agaaaggtag
     3241 aagaccccaa ggactttcct tcagaattgc taagtttttt gagtcatgct gtgtttagta
     3301 atagaactct tgcttgcttt gctatttaca ccacaaagga aaaagctgca ctgctataca
     3361 agaaaattat ggaaaaatat ttgatgtata gtgccttgac tagagatcat aatcagccat
     3421 accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg
     3481 aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac
     3541 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     3601 tgtggtttgt ccaaactcat caatgtatct taataacttc gtataatgta tgctatacga
     3661 agttatcgta cgggatctaa ttcaattaga gactaattca attagagcta attcaattag
     3721 gatccaagct tatcgatttc gaaccctcga ccgccggagt ataaatagag gcgcttcgtc
     3781 tacggagcga caattcaatt caaacaagca aagtgaacac gtcgctaagc gaaagctaag
     3841 caaataaaca agcgcagctg aacaagctaa acaatcggct cgaagccggt cgccaccatg
     3901 gcctcctccg aggacgtcat caaggagttc atgcgcttca aggtgcgcat ggagggctcc
     3961 gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc
     4021 cagaccgcca agctgaaggt gaccaagggc ggccccctgc ccttcgcctg ggacatcctg
     4081 tccccccagt tccagtacgg ctccaaggtg tacgtgaagc accccgccga catccccgac
     4141 tacaagaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac
     4201 ggcggcgtgg tgaccgtgac ccaggactcc tccctccagg acggctcctt catctacaag
     4261 gtgaagttca tcggcgtgaa cttcccctcc gacggccccg taatgcagaa gaagactatg
     4321 ggctgggagg cgtccaccga gcgcctgtac ccccgcgacg gcgtgctgaa gggcgagatc
     4381 cacaaggccc tgaagctgaa ggacggcggc cactacctgg tggagttcaa gtccatctac
     4441 atggccaaga agcccgtgca gctgcccggc tactactacg tggactccaa gctggacatc
     4501 acctcccaca acgaggacta caccatcgtg gagcagtacg agcgcgccga gggccgccac
     4561 cacctgttcc tgtaggggcc gcgactctag atcataatca gccataccac atttgtagag
     4621 gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat
     4681 gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     4741 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     4801 ctcatcaatg tatcttaacc gtctgtaagc agcgaaggcc catacgataa cttcgtataa
     4861 tgtatgctat acgaagttat gagctccagc ttttgttccc tttagtgagg gttaattgcg
     4921 cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     4981 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     5041 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     5101 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     5161 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     5221 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     5281 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     5341 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     5401 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     5461 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     5521 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     5581 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     5641 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     5701 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     5761 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     5821 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     5881 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     5941 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     6001 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     6061 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     6121 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     6181 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     6241 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     6301 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     6361 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     6421 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     6481 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     6541 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     6601 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     6661 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     6721 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     6781 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     6841 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     6901 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     6961 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     7021 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     7081 gtgccacctg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     7141 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     7201 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     7261 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     7321 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     7381 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     7441 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaaatg agctgattta
     7501 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttcca ttcgccattc
     7561 aggctgcgca actgttggga agggcgatcg gtgcgg
//
