LOCUS       sequence-314575-        6173 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR knock-in donor construct with fluorescent reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6173)
  AUTHORS   Chen YJ, Cheng YY, Wang W, Tian XJ, Lefever DE, Taft DA, Zhang J, 
            Xing J
  TITLE     Rapid, modular, and cost-effective generation of donor DNA 
            constructs for CRISPR-based gene knock-in.
  JOURNAL   Biol Methods Protoc. 2020 Mar 20;5(1):bpaa006. doi: 
            10.1093/biomethods/bpaa006. eCollection 2020.
  PUBMED    32411820
REFERENCE   2  (bases 1 to 6173)
  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: "Biol 
            Methods Protoc."; date: "2020-03-20"; volume: "5(1):bpaa006. doi"; 
            pages: " 10.1093/biomethods/bpaa006. eCollection 2020"
COMMENT     Sequence Label: pcDNA3-P1-N-mCherry-C
FEATURES             Location/Qualifiers
     source          1..6173
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             782..1489
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(927..945)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1195..1214
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1530..1547)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1536..1760
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1806..2234
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1893..1912)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2103..2124
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2243..2263)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2248..2577
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2428..2563
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2490..2509
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2644..3438
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2698..2717)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3308..3327
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3612..3733
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3649..3668)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3703..3722
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3782..3798)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3782..3798)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3795..3817)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3806..3822
                     /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(3830..3836,3837..3854,3855..3860))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3875..3896
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4013..4030)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4184..4772)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4264..4283)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4943..5734,5735..5803))
                     /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     5566..5585
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5804..5908)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5983..6002)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 ggtaccgagc tcggatccac tagtaacggc cgccagtgtg ctggaattct gctcgggccc
      721 ttctagacaa tcgatcaccg gtgctagcat cgaattccct agaggcggcg gcggcgtcga
      781 gatggtgagc aagggcgagg aggataacat ggccatcatc aaggagttca tgcgcttcaa
      841 ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg
      901 ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc
      961 cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct acgtgaagca
     1021 ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg
     1081 cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga
     1141 cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt
     1201 aatgcagaag aagaccatgg gctgggaggc ctcctccgag cggatgtacc ccgaggacgg
     1261 cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc actacgacgc
     1321 tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt
     1381 caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga
     1441 acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagg gagtctaact
     1501 cgagtgttaa cggcggccgg gatatcatgc ctcgactgtg ccttctagtt gccagccatc
     1561 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     1621 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     1681 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     1741 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
     1801 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     1861 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     1921 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     1981 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     2041 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     2101 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     2161 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     2221 atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     2281 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     2341 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2401 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     2461 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc
     2521 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     2581 ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt
     2641 cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     2701 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     2761 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     2821 ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     2881 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     2941 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     3001 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     3061 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     3121 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc
     3181 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     3241 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     3301 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     3361 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     3421 cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca
     3481 acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     3541 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     3601 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     3661 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     3721 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     3781 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     3841 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     3901 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     3961 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     4021 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     4081 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     4141 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4201 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     4261 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4321 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     4381 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     4441 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4501 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     4561 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     4621 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     4681 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     4741 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     4801 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     4861 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     4921 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     4981 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     5041 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     5101 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     5161 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     5221 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     5281 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     5341 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     5401 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     5461 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     5521 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     5581 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     5641 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     5701 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     5761 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     5821 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     5881 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
     5941 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc
     6001 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc
     6061 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct
     6121 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gtt
//
