LOCUS       sequence-249123-        5667 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPaint universal donor plasmid for gene exon insertion. Encodes 
            T2A-sfGFP-SV40 and 3xP3-RFP visible eye marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5667)
  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 5667)
  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-sfGFP-3xP3-RFP
FEATURES             Location/Qualifiers
     source          1..5667
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     51..73
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     65..82
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     66..82
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     92..111
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        92..110
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             203..256
                     /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          1069..1134
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             1264..1284
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    1556..1677
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    1678..1711
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      1937..1946
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1943..2620
                     /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(2073..2091)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(2123..2143)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     2341..2360
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2535..2558
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    2741..2862
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    2892..2925
                     /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(2944..2964)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2944..2962)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2983..2999)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2983..2999)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2996..3018)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3007..3023
                     /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(3031..3037,3038..3055,3056..3061))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3076..3097
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3214..3231)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3385..3973)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3465..3484)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4144..4935,4936..5004))
                     /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     4767..4786
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5005..5109)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5136..5592
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5223..5242)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5433..5454
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg
       61 acgttgtaaa acgacggcca gtgagcgcgc gtaatacgac tcactatagg gcgaattggg
      121 tacaagcttc acgacctgag gcgcgccggg ccagtaccca aaaagcgggg ggtccggtgg
      181 aagcggaggt agcgcggccg ccgagggccg cggcagcctg ctgacctgcg gcgatgtgga
      241 ggagaacccc gggcccgcta gcgtgtccaa gggcgaggag ctgttcaccg gcgtggtgcc
      301 catcctggtg gagctggatg gcgacgtgaa cggccacaag ttcagcgtgc gcggcgaggg
      361 cgagggcgac gccaccaacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
      421 gcccgtgccc tggcccaccc tggtgaccac cctgacctac ggcgtgcagt gcttcagccg
      481 ctaccccgat cacatgaagc agcacgattt cttcaagagc gccatgcccg agggctacgt
      541 gcaggagcgc accatcagct tcaaggatga cggcacctac aagacccgcg ccgaggtgaa
      601 gttcgagggc gataccctgg tgaaccgcat cgagctgaag ggcatcgatt tcaaggagga
      661 tggcaacatc ctgggccaca agctggagta caacttcaac agccacaacg tgtacatcac
      721 cgccgataag cagaagaacg gcatcaaggc caacttcaag atccgccaca atgtggagga
      781 tggctccgtg cagctggccg atcactacca gcagaacacc cccatcggcg acggcccagt
      841 gctgctgccc gataaccact acctgagcac ccagagcgtg ctgtccaagg accccaacga
      901 gaagcgcgat cacatggtgc tgctggagtt cgtgaccgcc gccggcatca ccctgggcat
      961 ggatgagctg tacaagtagg gtacctctag aggatctttg tgaaggaacc ttacttctgt
     1021 ggtgtgacat aattggacaa actacctaca gagatttaaa gctctaaggt aaatataaaa
     1081 tttttaagtg tataatgtgt taaactactg attctaattg tttgtgtatt ttagattcca
     1141 acctatggaa ctgatgaatg ggagcagtgg tggaatgcct ttaatgagga aaacctgttt
     1201 tgctcagaag aaatgccatc tagtgatgat gaggctactg ctgactctca acattctact
     1261 cctccaaaaa agaagagaaa ggtagaagac cccaaggact ttccttcaga attgctaagt
     1321 tttttgagtc atgctgtgtt tagtaataga actcttgctt gctttgctat ttacaccaca
     1381 aaggaaaaag ctgcactgct atacaagaaa attatggaaa aatatttgat gtatagtgcc
     1441 ttgactagag atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa
     1501 cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt
     1561 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     1621 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaata
     1681 acttcgtata atgtatgcta tacgaagtta tcgtacggga tctaattcaa ttagagacta
     1741 attcaattag agctaattca attaggatcc aagcttatcg atttcgaacc ctcgaccgcc
     1801 ggagtataaa tagaggcgct tcgtctacgg agcgacaatt caattcaaac aagcaaagtg
     1861 aacacgtcgc taagcgaaag ctaagcaaat aaacaagcgc agctgaacaa gctaaacaat
     1921 cggctcgaag ccggtcgcca ccatggcctc ctccgaggac gtcatcaagg agttcatgcg
     1981 cttcaaggtg cgcatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg
     2041 cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca agggcggccc
     2101 cctgcccttc gcctgggaca tcctgtcccc ccagttccag tacggctcca aggtgtacgt
     2161 gaagcacccc gccgacatcc ccgactacaa gaagctgtcc ttccccgagg gcttcaagtg
     2221 ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct
     2281 ccaggacggc tccttcatct acaaggtgaa gttcatcggc gtgaacttcc cctccgacgg
     2341 ccccgtaatg cagaagaaga ctatgggctg ggaggcgtcc accgagcgcc tgtacccccg
     2401 cgacggcgtg ctgaagggcg agatccacaa ggccctgaag ctgaaggacg gcggccacta
     2461 cctggtggag ttcaagtcca tctacatggc caagaagccc gtgcagctgc ccggctacta
     2521 ctacgtggac tccaagctgg acatcacctc ccacaacgag gactacacca tcgtggagca
     2581 gtacgagcgc gccgagggcc gccaccacct gttcctgtag gggccgcgac tctagatcat
     2641 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc
     2701 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta
     2761 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     2821 gcattctagt tgtggtttgt ccaaactcat caatgtatct taaccgtctg taagcagcga
     2881 aggcccatac gataacttcg tataatgtat gctatacgaa gttatgagct ccagcttttg
     2941 ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt
     3001 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     3061 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     3121 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     3181 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     3241 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     3301 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     3361 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     3421 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     3481 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     3541 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     3601 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     3661 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     3721 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     3781 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     3841 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     3901 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     3961 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     4021 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     4081 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     4141 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     4201 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     4261 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     4321 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     4381 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     4441 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     4501 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     4561 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     4621 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     4681 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     4741 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     4801 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     4861 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     4921 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     4981 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     5041 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     5101 ggttccgcgc acatttcccc gaaaagtgcc acctgacgcg ccctgtagcg gcgcattaag
     5161 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     5221 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     5281 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     5341 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     5401 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     5461 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     5521 ttggttaaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     5581 cgcttacaat ttccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     5641 ggcctcttcg ctattacgcc agctggc
//
