LOCUS       sequence-247975-        6003 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPaint universal donor plasmid for gene exon insertion. Encodes 
            T2A-QF2-SV40 and 3xP3-RFP visible eye marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6003)
  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 6003)
  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-QF2-3xP3-RFP
FEATURES             Location/Qualifiers
     source          1..6003
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     107..129
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     121..138
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     122..138
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     148..167
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        148..166
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             259..312
                     /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          1461..1526
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             1656..1676
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    1948..2069
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    2070..2103
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      2329..2338
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2335..3012
                     /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(2465..2483)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(2515..2535)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     2733..2752
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2927..2950
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    3133..3254
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    3284..3317
                     /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(3336..3356)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3336..3354)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3375..3391)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3375..3391)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3388..3410)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3399..3415
                     /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(3423..3429,3430..3447,3448..3453))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3468..3489
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3606..3623)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3777..4365)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3857..3876)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4536..5327,5328..5396))
                     /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     5159..5178
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5397..5501)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5528..5984
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5615..5634)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5825..5846
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa
       61 gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt
      121 tgtaaaacga cggccagtga gcgcgcgtaa tacgactcac tatagggcga attgggtaca
      181 agcttcacga cctgaggcgc gccgggccag tacccaaaaa gcggggggtc cggtggaagc
      241 ggaggtagcg cggccgccga gggccgcggc agcctgctga cctgcggcga tgtggaggag
      301 aaccccgggc ccgctagcat gccacccaag cgcaaaacgc ttaacgctgc ggctgaggct
      361 aacgctcatg ccgacggaca cgccgacgga aacgccgacg gacacgtggc caatacggcc
      421 gcgtcctcga ataatgcgag gttcgctgat ctcactaaca tcgatactcc gggtctggga
      481 cccacaacta cgaccctgct cgtggaacca gcacgctcaa agcgtcaacg agtgtcccgc
      541 gcatgcgacc agtgccgtgc agcccgagag aaatgcgacg gaatacagcc tgcgtgtttc
      601 ccgtgcgttt cccagggaag gtcctgcact tatcaggctt cgccgaaaaa gaggggagtt
      661 caaaccggtt atattcgtac gctggagctc gccctcgcct ggatgtttga aaatgtcgcg
      721 cgttccgaag atgccttgca taacctcctc gtccgtgacg ccggacaagg atcagctctg
      781 ctcgttggta aagattcgcc ggctgccgag cgactccatg cccgttgggc tactagccgt
      841 gtcaataaga gcattacccg cctcctccgt cagttggagc tccctcctac cgccacggct
      901 acggcctcga taatgccgca cgtgatggag cagcctctca gtaccagcat taaccccgtc
      961 aacgaccgct tcaacggtat tcccaacccc actccgtata actccgatgc agctctcgat
     1021 gctatcactc agaccaacga ttatggaagc gtaaatacac atggtatcct ctctacttac
     1081 ccgccaccgg ctacgcacct taatgaagct tccgtcgctc tcgctcccgg tggcgccccc
     1141 ccccgaccgc ctcctccgta tgttgacagc acgaccaatc acccgccgta ccactcgaat
     1201 ctggttccaa tggcgaactt tggttactcg accgttgatt acgatgccat ggttgacgat
     1261 ttggctagca ttgaatacac ggacgctgtg gatgtcgacc cacagtttat gaccaatctg
     1321 ggattcgttc ctggatgtaa cttctccgac attaatacat acgaacagtg aggtacctct
     1381 agaggatctt tgtgaaggaa ccttacttct gtggtgtgac ataattggac aaactaccta
     1441 cagagattta aagctctaag gtaaatataa aatttttaag tgtataatgt gttaaactac
     1501 tgattctaat tgtttgtgta ttttagattc caacctatgg aactgatgaa tgggagcagt
     1561 ggtggaatgc ctttaatgag gaaaacctgt tttgctcaga agaaatgcca tctagtgatg
     1621 atgaggctac tgctgactct caacattcta ctcctccaaa aaagaagaga aaggtagaag
     1681 accccaagga ctttccttca gaattgctaa gttttttgag tcatgctgtg tttagtaata
     1741 gaactcttgc ttgctttgct atttacacca caaaggaaaa agctgcactg ctatacaaga
     1801 aaattatgga aaaatatttg atgtatagtg ccttgactag agatcataat cagccatacc
     1861 acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa
     1921 cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa
     1981 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     2041 ggtttgtcca aactcatcaa tgtatcttaa taacttcgta taatgtatgc tatacgaagt
     2101 tatcgtacgg gatctaattc aattagagac taattcaatt agagctaatt caattaggat
     2161 ccaagcttat cgatttcgaa ccctcgaccg ccggagtata aatagaggcg cttcgtctac
     2221 ggagcgacaa ttcaattcaa acaagcaaag tgaacacgtc gctaagcgaa agctaagcaa
     2281 ataaacaagc gcagctgaac aagctaaaca atcggctcga agccggtcgc caccatggcc
     2341 tcctccgagg acgtcatcaa ggagttcatg cgcttcaagg tgcgcatgga gggctccgtg
     2401 aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag
     2461 accgccaagc tgaaggtgac caagggcggc cccctgccct tcgcctggga catcctgtcc
     2521 ccccagttcc agtacggctc caaggtgtac gtgaagcacc ccgccgacat ccccgactac
     2581 aagaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc
     2641 ggcgtggtga ccgtgaccca ggactcctcc ctccaggacg gctccttcat ctacaaggtg
     2701 aagttcatcg gcgtgaactt cccctccgac ggccccgtaa tgcagaagaa gactatgggc
     2761 tgggaggcgt ccaccgagcg cctgtacccc cgcgacggcg tgctgaaggg cgagatccac
     2821 aaggccctga agctgaagga cggcggccac tacctggtgg agttcaagtc catctacatg
     2881 gccaagaagc ccgtgcagct gcccggctac tactacgtgg actccaagct ggacatcacc
     2941 tcccacaacg aggactacac catcgtggag cagtacgagc gcgccgaggg ccgccaccac
     3001 ctgttcctgt aggggccgcg actctagatc ataatcagcc ataccacatt tgtagaggtt
     3061 ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca
     3121 attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     3181 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     3241 atcaatgtat cttaaccgtc tgtaagcagc gaaggcccat acgataactt cgtataatgt
     3301 atgctatacg aagttatgag ctccagcttt tgttcccttt agtgagggtt aattgcgcgc
     3361 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     3421 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     3481 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     3541 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     3601 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     3661 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     3721 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     3781 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     3841 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     3901 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     3961 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4021 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4081 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4141 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4201 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4261 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4321 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4381 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4441 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     4501 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     4561 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     4621 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     4681 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     4741 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     4801 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     4861 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     4921 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     4981 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     5041 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     5101 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     5161 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     5221 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     5281 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     5341 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     5401 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     5461 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     5521 ccacctgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     5581 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     5641 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     5701 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     5761 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     5821 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     5881 gatttataag ggattttgcc gatttcggcc tattggttaa aaaaatgagc tgatttaaca
     5941 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttccattc gccattcagg
     6001 ctg
//
