LOCUS       sequence-249127-        7803 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPaint universal donor plasmid for gene exon insertion. Encodes 
            T2A-LexGAD-SV40 and 3xP3-RFP visible eye marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7803)
  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 7803)
  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-LexGAD-3xP3-RFP
FEATURES             Location/Qualifiers
     source          1..7803
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     103..125
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     117..134
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     118..134
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     144..163
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        144..162
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             255..308
                     /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             321..341
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(351..602,603..956)
                     /codon_start=1
                     /gene="lexA"
                     /product="E. coli DNA-binding protein that represses genes 
                     involved in the SOS response to DNA damage"
                     /label=LexA
                     /note="The N-terminal DNA-binding domain recognizes the 
                     16-bp palindromic sequence 5'-CTGTATATATATACAG-3'."
                     /translation="MKALTARQQEVFDLIRDHISQTGMPPTRAEIAQRLGFRSPNAAEE
                     HLKALARKGVIEIVSGASRGIRLLQEEEEGLPLVGRVAAGEPLLAQQHIEGHYQVDPSL
                     FKPNADFLLRVSGMSMKDIGIMDGDLLAVHKTQDVRNGQVVVARIDDEVTVKRLKKQGN
                     KVELLPENSEFKPIVVDLRQQSFTIEGLAVGVIRNGDWL"
     intron          3257..3322
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             3452..3472
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3744..3865
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    3866..3899
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      4125..4134
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4131..4808
                     /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(4261..4279)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(4311..4331)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     4529..4548
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4723..4746
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    4929..5050
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    5080..5113
                     /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(5132..5152)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5132..5150)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5171..5187)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5171..5187)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5184..5206)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5195..5211
                     /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(5219..5225,5226..5243,5244..5249))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5264..5285
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5402..5419)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5573..6161)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5653..5672)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6332..7123,7124..7192))
                     /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     6955..6974
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7193..7297)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      7324..7780
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7411..7430)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7621..7642
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
       61 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
      121 aaacgacggc cagtgagcgc gcgtaatacg actcactata gggcgaattg ggtacaagct
      181 tcacgacctg aggcgcgccg ggccagtacc caaaaagcgg ggggtccggt ggaagcggag
      241 gtagcgcggc cgccgagggc cgcggcagcc tgctgacctg cggcgatgtg gaggagaacc
      301 ccgggcccgc tagcatgcca cccaagaaga agcgaaaagt agaagatcca atgaaggctc
      361 tcacggcccg acaacaggaa gtttttgatt tgatacggga tcatatatcc caaacgggta
      421 tgcctccgac ccgcgcagag atagcacagc gactgggctt tcgatcgcct aacgccgcgg
      481 aggagcactt gaaggcactg gcccgcaagg gtgtcattga aatcgtgtcc ggtgcgagcc
      541 gcggaatccg gctgttgcag gaggaggaag agggcctgcc actggtggga cgcgtggccg
      601 ctggcgagcc gctgctggcc cagcaacaca tagagggaca ctatcaggtg gacccctcct
      661 tgtttaagcc aaatgctgat ttcctgttgc gggtgtcggg aatgtccatg aaggacatcg
      721 gtattatgga tggtgacctc ctcgccgtcc ataagacaca ggatgtcagg aacggccagg
      781 tagtcgttgc caggatagac gatgaggtca ctgtgaaacg tctcaagaag caaggcaata
      841 aggtcgagct gctgccggag aatagcgagt tcaagccgat cgtggtggat ctgcgacagc
      901 agtcctttac tatcgagggc ttggccgtgg gtgtgatccg caacggagat tggctgggat
      961 ccatcgacag tgcggcccac cacgacaaca gcaccattcc actggacttc atgcctcgtg
     1021 acgcactgca tggatttgac tggagcgagg aggatgatat gagcgacgga ctcccattcc
     1081 tgaagaccga ccccaataac aatggtttct ttggcgacgg cagcctgctg tgtatcctgc
     1141 gaagtattgg ttttaagccg gaaaattaca cgaacagtaa tgtcaatcgt cttcccacaa
     1201 tgatcaccga ccgttacacg ctggccagtc gcagtactac aagccgcctg ctgcaaagct
     1261 atcttaacaa cttccacccc tactgcccca tagtgcatag tcctactctc atgatgcttt
     1321 acaacaatca gattgagata gcatcgaagg accagtggca gatcctcttc aactgcattc
     1381 tcgccatcgg cgcttggtgt attgaaggcg agagcacaga tatcgacgtg ttttactatc
     1441 aaaacgccaa gtcgcacctc acctcgaagg tcttcgaaag tggaagcatt atcctcgtga
     1501 cagccctcca tctgctgagt cgttataccc agtggcgtca gaaaactaat acatcctaca
     1561 actttcattc gttcagcatt cggatggcga ttagcctggg acttaatcgt gacctcccaa
     1621 gtagcttcag cgattcgtcg atcctggaac agcgccggag gatttggtgg agtgtgtaca
     1681 gctgggagat ccagctgtcc ctcttgtacg gacgcagcat tcagctgagt cagaacacga
     1741 tttccttccc gtccagcgtg gacgacgttc aacggacaac tacgggcccg accatctacc
     1801 acggtatcat tgagacggcc cgcttgcttc aagtgtttac gaagatttat gagctggata
     1861 agacagtgac cgcggagaag agtccgatct gcgccaagaa atgcctgatg atctgcaatg
     1921 agatagagga ggtgagccgc caggcaccca agttcctgca gatggacata agcactacgg
     1981 cgctgactaa tctcctcaaa gaacacccct ggctgagttt cacccgcttc gagctgaagt
     2041 ggaaacagct gagtctgatt atatacgttt tgcgggactt tttcacaaat tttacgcaga
     2101 agaagtcgca gcttgagcaa gatcagaacg accatcagtc ctacgaggta aagaggtgca
     2161 gcataatgct ctccgacgca gcccagcgca ccgtgatgtc ggtgtcgtcc tatatggaca
     2221 accataatgt gaccccttat ttcgcctgga attgctccta ctatttgttc aacgccgtgt
     2281 tggtcccaat caagactctt ctgagcaact ccaaatcgaa cgccgagaat aacgagacag
     2341 cacagctctt gcagcaaatt aacaccgtgt tgatgctgtt gaagaaactc gctacattca
     2401 agattcagac ttgcgaaaag tacatccagg tgttggaaga ggtgtgcgcc ccattccttc
     2461 tttcgcaatg cgcaattccc ctcccgcaca taagttacaa taattccaac ggctccgcta
     2521 tcaagaatat cgtcggctcc gctacaatcg cgcagtatcc cacgctgcct gaggaaaatg
     2581 ttaacaacat atcggtgaag tatgtaagtc caggcagcgt cggcccgagc cctgtgccgc
     2641 tgaaaagcgg agcctccttc agcgatctgg tcaagctgct cagcaaccgc ccaccatccc
     2701 gcaactcccc tgtcactata ccccgatcga ccccgtcgca tcgtagcgtt accccattcc
     2761 tgggacaaca gcagcaactg cagagtctgg tgccacttac gccctcggca ttgtttggcg
     2821 gagccaactt caaccagagt ggtaacattg cggacagctc cttgagcttc acgtttacca
     2881 acagctccaa cggtccaaac ttgatcacga ctcagacaaa ctcccaggca ttgtcccagc
     2941 ccattgccag ctcgaatgtg catgataact ttatgaataa tgaaattact gcctccaaga
     3001 ttgacgacgg caacaacagc aagccccttt ccccaggctg gactgaccag acggcctaca
     3061 atgctttcgg tatcaccact ggcatgttca acaccacaac gatggacgac gtatataact
     3121 atctttttga tgatgaggat actccaccga acccaaagaa ggagtagggt acctctagag
     3181 gatctttgtg aaggaacctt acttctgtgg tgtgacataa ttggacaaac tacctacaga
     3241 gatttaaagc tctaaggtaa atataaaatt tttaagtgta taatgtgtta aactactgat
     3301 tctaattgtt tgtgtatttt agattccaac ctatggaact gatgaatggg agcagtggtg
     3361 gaatgccttt aatgaggaaa acctgttttg ctcagaagaa atgccatcta gtgatgatga
     3421 ggctactgct gactctcaac attctactcc tccaaaaaag aagagaaagg tagaagaccc
     3481 caaggacttt ccttcagaat tgctaagttt tttgagtcat gctgtgttta gtaatagaac
     3541 tcttgcttgc tttgctattt acaccacaaa ggaaaaagct gcactgctat acaagaaaat
     3601 tatggaaaaa tatttgatgt atagtgcctt gactagagat cataatcagc cataccacat
     3661 ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata
     3721 aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa
     3781 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     3841 tgtccaaact catcaatgta tcttaataac ttcgtataat gtatgctata cgaagttatc
     3901 gtacgggatc taattcaatt agagactaat tcaattagag ctaattcaat taggatccaa
     3961 gcttatcgat ttcgaaccct cgaccgccgg agtataaata gaggcgcttc gtctacggag
     4021 cgacaattca attcaaacaa gcaaagtgaa cacgtcgcta agcgaaagct aagcaaataa
     4081 acaagcgcag ctgaacaagc taaacaatcg gctcgaagcc ggtcgccacc atggcctcct
     4141 ccgaggacgt catcaaggag ttcatgcgct tcaaggtgcg catggagggc tccgtgaacg
     4201 gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg
     4261 ccaagctgaa ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc ctgtcccccc
     4321 agttccagta cggctccaag gtgtacgtga agcaccccgc cgacatcccc gactacaaga
     4381 agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg
     4441 tggtgaccgt gacccaggac tcctccctcc aggacggctc cttcatctac aaggtgaagt
     4501 tcatcggcgt gaacttcccc tccgacggcc ccgtaatgca gaagaagact atgggctggg
     4561 aggcgtccac cgagcgcctg tacccccgcg acggcgtgct gaagggcgag atccacaagg
     4621 ccctgaagct gaaggacggc ggccactacc tggtggagtt caagtccatc tacatggcca
     4681 agaagcccgt gcagctgccc ggctactact acgtggactc caagctggac atcacctccc
     4741 acaacgagga ctacaccatc gtggagcagt acgagcgcgc cgagggccgc caccacctgt
     4801 tcctgtaggg gccgcgactc tagatcataa tcagccatac cacatttgta gaggttttac
     4861 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg
     4921 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     4981 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     5041 atgtatctta accgtctgta agcagcgaag gcccatacga taacttcgta taatgtatgc
     5101 tatacgaagt tatgagctcc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg
     5161 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     5221 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     5281 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     5341 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     5401 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     5461 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     5521 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     5581 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5641 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     5701 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5761 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5821 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5881 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5941 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     6001 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     6061 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     6121 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     6181 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     6241 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     6301 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     6361 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     6421 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     6481 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     6541 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     6601 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     6661 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     6721 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     6781 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     6841 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     6901 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     6961 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     7021 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     7081 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     7141 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     7201 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     7261 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     7321 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     7381 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     7441 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     7501 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     7561 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     7621 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     7681 tataagggat tttgccgatt tcggcctatt ggttaaaaaa atgagctgat ttaacaaaaa
     7741 tttaacgcga attttaacaa aatattaacg cttacaattt ccattcgcca ttcaggctgc
     7801 gca
//
