LOCUS       sequence-373521-        4046 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector for generating dsDNA donors for homology-directed repair. 
            Contains the visible marker 3xP3-DsRed..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4046)
  AUTHORS   Gratz SJ, Ukken FP, Rubinstein CD, Thiede G, Donohue LK, Cummings 
            AM, O'Connor-Giles KM
  TITLE     Highly specific and efficient CRISPR/Cas9-catalyzed 
            homology-directed repair in Drosophila.
  JOURNAL   Genetics. 2014 Apr;196(4):961-71. doi: 10.1534/genetics.113.160713. 
            Epub 2014 Jan 29.
  PUBMED    24478335
REFERENCE   2  (bases 1 to 4046)
  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"; doi: 
            "10.1534/genetics.113.160713"; journalName: "Genetics"; date: 
            "2014-04"; volume: "196"; issue: "4"; pages: "961-71"
COMMENT     Sequence Label: pHD-DsRed
FEATURES             Location/Qualifiers
     source          1..4046
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    106..139
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      365..374
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             371..1048
                     /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(501..519)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(551..571)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     769..788
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     963..986
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    1169..1290
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1206..1225)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1260..1279
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    1297..1330
                     /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(1866..1883)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1866..1882)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1981..1998)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2152..2740)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2232..2251)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2862..3653,3654..3722))
                     /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     3485..3504
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3723..3814)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3856..3875
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3881..3900
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 aaagactggg cctttcgccc gggctaatta tggggtgtcg cccttcgctg aagcaggtgg
       61 aattcttgca tgctagcggc cgcggacata tgcacacctg cgatcataac ttcgtataat
      121 gtatgctata cgaagttatc gtacgggatc taattcaatt agagactaat tcaattagag
      181 ctaattcaat taggatccaa gcttatcgat ttcgaaccct cgaccgccgg agtataaata
      241 gaggcgcttc gtctacggag cgacaattca attcaaacaa gcaaagtgaa cacgtcgcta
      301 agcgaaagct aagcaaataa acaagcgcag ctgaacaagc taaacaatcg gctcgaagcc
      361 ggtcgccacc atggcctcct ccgaggacgt catcaaggag ttcatgcgct tcaaggtgcg
      421 catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc
      481 ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggcggccccc tgcccttcgc
      541 ctgggacatc ctgtcccccc agttccagta cggctccaag gtgtacgtga agcaccccgc
      601 cgacatcccc gactacaaga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat
      661 gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctcc aggacggctc
      721 cttcatctac aaggtgaagt tcatcggcgt gaacttcccc tccgacggcc ccgtaatgca
      781 gaagaagact atgggctggg aggcgtccac cgagcgcctg tacccccgcg acggcgtgct
      841 gaagggcgag atccacaagg ccctgaagct gaaggacggc ggccactacc tggtggagtt
      901 caagtccatc tacatggcca agaagcccgt gcagctgccc ggctactact acgtggactc
      961 caagctggac atcacctccc acaacgagga ctacaccatc gtggagcagt acgagcgcgc
     1021 cgagggccgc caccacctgt tcctgtaggg gccgcgactc tagatcataa tcagccatac
     1081 cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa
     1141 acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa
     1201 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     1261 tggtttgtcc aaactcatca atgtatctta accggtataa cttcgtataa tgtatgctat
     1321 acgaagttat agaagagcac tagtaaagat ctccatgcat aaggcgcgcc taggccttct
     1381 gcagctcgag gctcttccgt caatcgagtt caagggcgac acaaaattta ttctaaatgc
     1441 ataataaata ctgataacat cttatagttt gtattatatt ttgtattatc gttgacatgt
     1501 ataattttga tatcaaaaac tgattttccc tttattattt tcgagattta ttttcttaat
     1561 tctctttaac aaactagaaa tattgtatat acaaaaaatc ataaataata gatgaatagt
     1621 ttaattatag gtgttcatca atcgaaaaag caacgtatct tatttaaagt gcgttgcttt
     1681 tttctcattt ataaggttaa ataattctca tatatcaagc aaagtgacag gcgcccttaa
     1741 atattctgac aaatgctctt tccctaaact ccccccataa aaaaacccgc cgaagcgggt
     1801 ttttacgtta tttgcggatt aacgattact cgttatcaga accgcccagg gggcccgagc
     1861 ttaagactgg ccgtcgtttt acaacacaga aagagtttgt agaaacgcaa aaaggccatc
     1921 cgtcaggggc cttctgctta gtttgatgcc tggcagttcc ctactctcgc cttccgcttc
     1981 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     2041 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     2101 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     2161 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     2221 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     2281 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     2341 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     2401 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     2461 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     2521 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggg ctaactacgg
     2581 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     2641 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     2701 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     2761 tacggggtct gacgctcagt ggaacgacgc gcgcgtaact cacgttaagg gattttggtc
     2821 atgagcttgc gccgtcccgt caagtcagcg taatgctctg cttaccaatg cttaatcagt
     2881 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     2941 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagcgctgc gatgataccg
     3001 cgagaaccac gctcaccggc tccggattta tcagcaataa accagccagc cggaagggcc
     3061 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     3121 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc catcgctaca
     3181 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     3241 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     3301 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     3361 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     3421 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     3481 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     3541 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     3601 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     3661 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     3721 atattcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     3781 tacatatttg aatgtattta gaaaaataaa caaatagggg tcagtgttac aaccaattaa
     3841 ccaattctga acattatcgc gagcccattt atacctgaat atggctcata acaccccttg
     3901 tttgcctggc ggcagtagcg cggtggtccc acctgacccc atgccgaact cagaagtgaa
     3961 acgccgtagc gccgatggta gtgtggggac tccccatgcg agagtaggga actgccaggc
     4021 atcaaataaa acgaaaggct cagtcg
//
