LOCUS       sequence-254075-        4231 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPaint universal donor plasmid for gene exon insertion. Encodes 
            3xP3-RFP visible eye marker and multiple cloning site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4231)
  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 4231)
  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-3xP3-RFP
FEATURES             Location/Qualifiers
     source          1..4231
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     117..139
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     131..148
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     132..148
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     158..177
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        158..176
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    308..341
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      567..576
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             573..1250
                     /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(703..721)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(753..773)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     971..990
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1165..1188
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    1371..1492
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1408..1427)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1462..1481
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    1522..1555
                     /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(1574..1594)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1574..1592)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1613..1629)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1613..1629)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1626..1648)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1637..1653
                     /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(1661..1667,1668..1685,1686..1691))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1706..1727
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1844..1861)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2015..2603)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2095..2114)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2774..3565,3566..3634))
                     /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     3397..3416
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3635..3739)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3766..4222
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3853..3872)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4063..4084
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca
       61 gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca
      121 gtcacgacgt tgtaaaacga cggccagtga gcgcgcgtaa tacgactcac tatagggcga
      181 attgggtaca agcttcacga cctgaggcgc gccgggccag tacccaaaaa gcggggggtc
      241 cggtggaagc ggaggtagcg cggccgcacg ctagctgctc gagacggtac ctagaattca
      301 cagatctata acttcgtata atgtatgcta tacgaagtta tcgtacggga tctaattcaa
      361 ttagagacta attcaattag agctaattca attaggatcc aagcttatcg atttcgaacc
      421 ctcgaccgcc ggagtataaa tagaggcgct tcgtctacgg agcgacaatt caattcaaac
      481 aagcaaagtg aacacgtcgc taagcgaaag ctaagcaaat aaacaagcgc agctgaacaa
      541 gctaaacaat cggctcgaag ccggtcgcca ccatggcctc ctccgaggac gtcatcaagg
      601 agttcatgcg cttcaaggtg cgcatggagg gctccgtgaa cggccacgag ttcgagatcg
      661 agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca
      721 agggcggccc cctgcccttc gcctgggaca tcctgtcccc ccagttccag tacggctcca
      781 aggtgtacgt gaagcacccc gccgacatcc ccgactacaa gaagctgtcc ttccccgagg
      841 gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg
      901 actcctccct ccaggacggc tccttcatct acaaggtgaa gttcatcggc gtgaacttcc
      961 cctccgacgg ccccgtaatg cagaagaaga ctatgggctg ggaggcgtcc accgagcgcc
     1021 tgtacccccg cgacggcgtg ctgaagggcg agatccacaa ggccctgaag ctgaaggacg
     1081 gcggccacta cctggtggag ttcaagtcca tctacatggc caagaagccc gtgcagctgc
     1141 ccggctacta ctacgtggac tccaagctgg acatcacctc ccacaacgag gactacacca
     1201 tcgtggagca gtacgagcgc gccgagggcc gccaccacct gttcctgtag gggccgcgac
     1261 tctagatcat aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc
     1321 cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta
     1381 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     1441 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct taaccgtctg
     1501 taagcagcga aggcccatac gataacttcg tataatgtat gctatacgaa gttatgagct
     1561 ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc
     1621 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     1681 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     1741 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     1801 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     1861 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     1921 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     1981 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     2041 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     2101 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     2161 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     2221 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     2281 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     2341 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     2401 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     2461 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     2521 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     2581 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     2641 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     2701 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     2761 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     2821 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     2881 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     2941 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     3001 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     3061 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     3121 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     3181 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     3241 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     3301 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     3361 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     3421 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     3481 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     3541 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     3601 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     3661 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     3721 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgcg ccctgtagcg
     3781 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     3841 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     3901 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     3961 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     4021 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     4081 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     4141 tttcggccta ttggttaaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     4201 aaaatattaa cgcttacaat ttccattcgc c
//
