LOCUS       sequence-248019-        6009 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPaint universal donor plasmid for gene exon insertion. Encodes 
            T2A-p65-SV40 and 3xP3-RFP visible eye marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6009)
  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 6009)
  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-p65-3xP3-RFP
FEATURES             Location/Qualifiers
     source          1..6009
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     115..137
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     129..146
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     130..146
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     156..175
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        156..174
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             267..320
                     /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             357..377
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             828..1199
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALLSQISS"
     intron          1475..1540
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             1670..1690
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    1962..2083
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    2084..2117
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      2343..2352
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2349..3026
                     /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(2479..2497)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(2529..2549)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     2747..2766
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2941..2964
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    3147..3268
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    3298..3331
                     /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(3350..3370)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3350..3368)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3389..3405)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3389..3405)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3402..3424)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3413..3429
                     /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(3437..3443,3444..3461,3462..3467))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3482..3503
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3620..3637)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3791..4379)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3871..3890)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4550..5341,5342..5410))
                     /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     5173..5192
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5411..5515)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5542..5998
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5629..5648)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5839..5860
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc
       61 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt
      121 cacgacgttg taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat
      181 tgggtacaag cttcacgacc tgaggcgcgc cgggccagta cccaaaaagc ggggggtccg
      241 gtggaagcgg aggtagcgcg gccgccgagg gccgcggcag cctgctgacc tgcggcgatg
      301 tggaggagaa ccccgggccc gctagcatgg ataaagcgga attaattccc gagcctccaa
      361 aaaagaagag aaaggtcgaa ttgggatcca cgccgatgga gttccagtac ctgcccgata
      421 cggatgaccg tcaccgtatc gaagaaaagc ggaagcgaac ctatgaaacc ttcaagtcca
      481 tcatgaaaaa gtcccccttc tcgggcccca cggacccgcg ccccccgccc cgtcgtattg
      541 cggttccttc gcgcagcagt gccagcgtcc ccaaacccgc accgcagccc tacccgttca
      601 cttcctccct tagcacgatt aactatgatg agttccccac gatggtgttc cccagtggac
      661 aaatttccca ggcatcggca ctggctccgg ccccaccgca agtcctcccc caggcgcccg
      721 ctccggcacc ggctcccgca atggtgagtg ctctggccca ggcccccgct ccagtccccg
      781 tgctggcgcc tggaccccca caggcagttg cccctcctgc tccgaaacca acgcaggcgg
      841 gcgaaggaac cctgagcgag gccctcttgc agcttcagtt cgatgacgaa gacttgggag
      901 ccctgctggg taacagcaca gaccctgccg tattcaccga tctcgcatcc gtggacaaca
      961 gcgagtttca gcagctcttg aatcagggaa tcccggtcgc acctcatacc acagagccca
     1021 tgctgatgga atacccggag gctatcacgc gactggtgac cggcgcacag cgaccacccg
     1081 atccagcccc tgccccactg ggtgccccgg gtttgcccaa tggcctcctc agcggcgatg
     1141 aggatttctc cagcatcgct gatatggatt tctccgcttt gctgagccag ataagctccg
     1201 ctagcggagg aggtggtgga ggtggaggtg gaggtactag tctggagatc gaggccgcct
     1261 tcctggagcg cgagaacacc gccctggaga cccgcgtggc cgagctgcgc cagcgcgtgc
     1321 agcgcctgcg caaccgcgtg agccagtacc gcacccgcta cggccccctg ggcggcggca
     1381 agtaaggtac ctctagagga tctttgtgaa ggaaccttac ttctgtggtg tgacataatt
     1441 ggacaaacta cctacagaga tttaaagctc taaggtaaat ataaaatttt taagtgtata
     1501 atgtgttaaa ctactgattc taattgtttg tgtattttag attccaacct atggaactga
     1561 tgaatgggag cagtggtgga atgcctttaa tgaggaaaac ctgttttgct cagaagaaat
     1621 gccatctagt gatgatgagg ctactgctga ctctcaacat tctactcctc caaaaaagaa
     1681 gagaaaggta gaagacccca aggactttcc ttcagaattg ctaagttttt tgagtcatgc
     1741 tgtgtttagt aatagaactc ttgcttgctt tgctatttac accacaaagg aaaaagctgc
     1801 actgctatac aagaaaatta tggaaaaata tttgatgtat agtgccttga ctagagatca
     1861 taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc
     1921 ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt
     1981 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     2041 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaataactt cgtataatgt
     2101 atgctatacg aagttatcgt acgggatcta attcaattag agactaattc aattagagct
     2161 aattcaatta ggatccaagc ttatcgattt cgaaccctcg accgccggag tataaataga
     2221 ggcgcttcgt ctacggagcg acaattcaat tcaaacaagc aaagtgaaca cgtcgctaag
     2281 cgaaagctaa gcaaataaac aagcgcagct gaacaagcta aacaatcggc tcgaagccgg
     2341 tcgccaccat ggcctcctcc gaggacgtca tcaaggagtt catgcgcttc aaggtgcgca
     2401 tggagggctc cgtgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct
     2461 acgagggcac ccagaccgcc aagctgaagg tgaccaaggg cggccccctg cccttcgcct
     2521 gggacatcct gtccccccag ttccagtacg gctccaaggt gtacgtgaag caccccgccg
     2581 acatccccga ctacaagaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga
     2641 acttcgagga cggcggcgtg gtgaccgtga cccaggactc ctccctccag gacggctcct
     2701 tcatctacaa ggtgaagttc atcggcgtga acttcccctc cgacggcccc gtaatgcaga
     2761 agaagactat gggctgggag gcgtccaccg agcgcctgta cccccgcgac ggcgtgctga
     2821 agggcgagat ccacaaggcc ctgaagctga aggacggcgg ccactacctg gtggagttca
     2881 agtccatcta catggccaag aagcccgtgc agctgcccgg ctactactac gtggactcca
     2941 agctggacat cacctcccac aacgaggact acaccatcgt ggagcagtac gagcgcgccg
     3001 agggccgcca ccacctgttc ctgtaggggc cgcgactcta gatcataatc agccatacca
     3061 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac
     3121 ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat
     3181 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     3241 gtttgtccaa actcatcaat gtatcttaac cgtctgtaag cagcgaaggc ccatacgata
     3301 acttcgtata atgtatgcta tacgaagtta tgagctccag cttttgttcc ctttagtgag
     3361 ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     3421 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     3481 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     3541 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     3601 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     3661 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     3721 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     3781 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     3841 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     3901 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     3961 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4021 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4081 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4141 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     4201 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     4261 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4321 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     4381 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     4441 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     4501 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     4561 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     4621 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     4681 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     4741 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     4801 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     4861 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     4921 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     4981 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     5041 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     5101 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     5161 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     5221 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     5281 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     5341 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     5401 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     5461 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     5521 ttccccgaaa agtgccacct gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     5581 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     5641 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     5701 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     5761 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     5821 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     5881 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaaat
     5941 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttcc
     6001 attcgccat
//
