LOCUS       sequence-248022-        6257 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPaint universal donor plasmid for gene exon insertion. Encodes 
            T2A-Gal80-SV40 and 3xP3-RFP visible eye marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6257)
  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 6257)
  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-Gal80-3xP3-RFP
FEATURES             Location/Qualifiers
     source          1..6257
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     23..45
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     37..54
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     38..54
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     64..83
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        64..82
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             175..228
                     /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             235..1542
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL80"
                     /product="transcriptional repressor that inhibits GAL4"
                     /label=GAL80
                     /note="GAL80 binds to and inhibits the transcriptional 
                     activation domain of GAL4."
                     /translation="MDYNKRSSVSTVPNAAPIRVGFVGLNAAKGWAIKTHYPAILQLSS
                     QFQITALYSPKIETSIATIQRLKLSNATAFPTLESFASSSTIDMIVIAIQVASHYEVVM
                     PLLEFSKNNPNLKYLFVEWALACSLDQAESIYKAAAERGVQTIISLQGRKSPYILRAKE
                     LISQGYIGDINSIEIAGNGGWYGYERPVKSPKYIYEIGNGVDLVTTTFGHTIDILQYMT
                     SSYFSRINAMVFNNIPEQELIDERGNRLGQRVPKTVPDHLLFQGTLLNGNVPVSCSFKG
                     GKPTKKFTKNLVIDIHGTKGDLKLEGDAGFAEISNLVLYYSGTRANDFPLANGQQAPLD
                     PGYDAGKEIMEVYHLRNYNAIVGNIHRLYQSISDFHFNTKKIPELPSQFVMQGFDFEGF
                     PTLMDALILHRLIESVYKSNMMGSTLNVSNISHYSL"
     intron          1631..1696
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             1826..1846
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2118..2239
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    2240..2273
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      2499..2508
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2505..3182
                     /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(2635..2653)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(2685..2705)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     2903..2922
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3097..3120
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    3303..3424
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    3454..3487
                     /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(3506..3526)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3506..3524)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3545..3561)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3545..3561)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3558..3580)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3569..3585
                     /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(3593..3599,3600..3617,3618..3623))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3638..3659
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3776..3793)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3947..4535)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4027..4046)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4706..5497,5498..5566))
                     /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     5329..5348
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5567..5671)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5698..6154
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5785..5804)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5995..6016
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgagcgc
       61 gcgtaatacg actcactata gggcgaattg ggtacaagct tcacgacctg aggcgcgccg
      121 ggccagtacc caaaaagcgg ggggtccggt ggaagcggag gtagcgcggc cgccgagggc
      181 cgcggcagcc tgctgacctg cggcgatgtg gaggagaacc ccgggcccgc tagcatggac
      241 tacaacaaga gatcttcggt ctcaaccgtg cctaatgcag ctcccataag agtcggattc
      301 gtcggtctca acgcagccaa aggatgggca atcaagacac attaccccgc catactgcaa
      361 ctatcgtcac aatttcaaat cactgcctta tacagtccaa aaattgagac ttctattgcc
      421 accattcagc gtctaaaatt gagtaatgcc actgcttttc ccactttaga gtcatttgca
      481 tcatcttcca ctatagatat gatagtgata gctatccaag tggccagcca ttatgaagtt
      541 gttatgcctc tcttggaatt ctccaaaaat aatccgaacc tcaagtatct tttcgtagaa
      601 tgggcccttg catgttcact agatcaagcc gaatccattt ataaggctgc tgctgaacgt
      661 ggggttcaaa ccatcatctc tttacaaggt cgtaaatcac catatatttt gagagcaaaa
      721 gaattaatat ctcaaggcta tatcggcgac attaattcga tcgagattgc tggaaatggc
      781 ggttggtacg gctacgaaag gcctgttaaa tcaccaaaat acatctatga aatcgggaac
      841 ggtgtagatc tggtaaccac aacatttggt cacacaatcg atattttaca atacatgaca
      901 agttcgtact tttccaggat aaatgcaatg gttttcaata atattccaga gcaagagctg
      961 atagatgagc gtggtaaccg attgggccag cgagtcccaa agacagtacc ggatcatctt
     1021 ttattccaag gcacattgtt aaatggcaat gttccagtgt catgcagttt caaaggtggc
     1081 aaacctacca aaaaatttac caaaaatttg gtcattgaca ttcacggtac caagggagat
     1141 ttgaaacttg aaggcgatgc cggcttcgca gaaatttcaa atctggtcct ttactacagt
     1201 ggaactagag caaacgactt cccgctagcc aatggacaac aagctccttt agacccgggg
     1261 tatgatgcag gtaaagaaat catggaagta tatcatttac gaaattataa tgccattgtg
     1321 ggtaatattc atcgactgta tcaatctatc tctgacttcc acttcaatac aaagaaaatt
     1381 cctgaattac cctcacaatt tgtaatgcaa ggtttcgatt tcgaaggctt tcccaccttg
     1441 atggatgctc tgatattaca caggttaatc gagagcgttt ataaaagtaa catgatgggc
     1501 tccacattaa acgttagcaa tatctcgcat tatagtttat aagtacctct agaggatctt
     1561 tgtgaaggaa ccttacttct gtggtgtgac ataattggac aaactaccta cagagattta
     1621 aagctctaag gtaaatataa aatttttaag tgtataatgt gttaaactac tgattctaat
     1681 tgtttgtgta ttttagattc caacctatgg aactgatgaa tgggagcagt ggtggaatgc
     1741 ctttaatgag gaaaacctgt tttgctcaga agaaatgcca tctagtgatg atgaggctac
     1801 tgctgactct caacattcta ctcctccaaa aaagaagaga aaggtagaag accccaagga
     1861 ctttccttca gaattgctaa gttttttgag tcatgctgtg tttagtaata gaactcttgc
     1921 ttgctttgct atttacacca caaaggaaaa agctgcactg ctatacaaga aaattatgga
     1981 aaaatatttg atgtatagtg ccttgactag agatcataat cagccatacc acatttgtag
     2041 aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga
     2101 atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     2161 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     2221 aactcatcaa tgtatcttaa taacttcgta taatgtatgc tatacgaagt tatcgtacgg
     2281 gatctaattc aattagagac taattcaatt agagctaatt caattaggat ccaagcttat
     2341 cgatttcgaa ccctcgaccg ccggagtata aatagaggcg cttcgtctac ggagcgacaa
     2401 ttcaattcaa acaagcaaag tgaacacgtc gctaagcgaa agctaagcaa ataaacaagc
     2461 gcagctgaac aagctaaaca atcggctcga agccggtcgc caccatggcc tcctccgagg
     2521 acgtcatcaa ggagttcatg cgcttcaagg tgcgcatgga gggctccgtg aacggccacg
     2581 agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc
     2641 tgaaggtgac caagggcggc cccctgccct tcgcctggga catcctgtcc ccccagttcc
     2701 agtacggctc caaggtgtac gtgaagcacc ccgccgacat ccccgactac aagaagctgt
     2761 ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga
     2821 ccgtgaccca ggactcctcc ctccaggacg gctccttcat ctacaaggtg aagttcatcg
     2881 gcgtgaactt cccctccgac ggccccgtaa tgcagaagaa gactatgggc tgggaggcgt
     2941 ccaccgagcg cctgtacccc cgcgacggcg tgctgaaggg cgagatccac aaggccctga
     3001 agctgaagga cggcggccac tacctggtgg agttcaagtc catctacatg gccaagaagc
     3061 ccgtgcagct gcccggctac tactacgtgg actccaagct ggacatcacc tcccacaacg
     3121 aggactacac catcgtggag cagtacgagc gcgccgaggg ccgccaccac ctgttcctgt
     3181 aggggccgcg actctagatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt
     3241 taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg
     3301 ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     3361 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     3421 cttaaccgtc tgtaagcagc gaaggcccat acgataactt cgtataatgt atgctatacg
     3481 aagttatgag ctccagcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat
     3541 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     3601 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     3661 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     3721 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     3781 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     3841 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     3901 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     3961 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4021 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4081 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4141 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4201 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4261 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4321 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     4381 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     4441 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     4501 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     4561 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     4621 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     4681 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     4741 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     4801 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     4861 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     4921 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     4981 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     5041 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     5101 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     5161 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     5221 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     5281 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     5341 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     5401 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     5461 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     5521 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     5581 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     5641 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     5701 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     5761 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     5821 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     5881 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     5941 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     6001 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     6061 ggattttgcc gatttcggcc tattggttaa aaaaatgagc tgatttaaca aaaatttaac
     6121 gcgaatttta acaaaatatt aacgcttaca atttccattc gccattcagg ctgcgcaact
     6181 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
     6241 gtgctgcaag gcgatta
//
