LOCUS       sequence-314573-        7994 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR knock-in donor construct with fluorescent reporter and 
            removable selectable marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7994)
  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 7994)
  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-miRFP670-C-LoxP-Neo
FEATURES             Location/Qualifiers
     source          1..7994
                     /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..1726
                     /codon_start=1
                     /product="bright monomeric near-infrared fluorescent 
                     protein engineered from a bacterial phytochrome, with an 
                     emission maximum of 670 nm (Shcherbakova et al., 2016)"
                     /label=miRFP670
                     /label=ORF frame 1
                     /translation="MVAGHASGSPAFGTASHSNSEHEEIHLAGSIQPHGALLVVSEHDH
                     RVIQASANAAEFLNLGSVLGVPLAEIDGDLLIKILPHLDPTAEGMPVAVRCRIGNPSTE
                     YCGLMHRPPEGGLIIELERAGPSIDLSGTLAPALERIRTAGSLRALCDDTVLLFQQCTG
                     YDRVMVYRFDEQGHGLVFSECHVPGLESYFGNRYPSSTVPQMARQLYVRQRVRVLVDVT
                     YQPVPLEPRLSPLTGRDLDMSGCFLRSMSPCHLQFLKDMGVRATLAVSLVVGGKLWGLV
                     VCHHYLPRFIRFELRAICKRLAERIATRITALES"
     protein_bind    complement(1763..1796)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1797..2125
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1976..2111
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2192..2986
                     /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"
     polyA_signal    3160..3281
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(3286..3319)
                     /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(3351..3368)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3357..3581
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3627..4055
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3714..3733)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3924..3945
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4069..4398
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4249..4384
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4465..5259
                     /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"
     polyA_signal    5433..5554
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5603..5619)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5603..5619)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5616..5638)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5627..5643
                     /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(5651..5657,5658..5675,5676..5681))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5696..5717
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5834..5851)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6005..6593)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6085..6104)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6764..7555,7556..7624))
                     /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     7387..7406
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7625..7729)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7804..7823)
                     /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 gatggtagca ggtcatgcct ctggcagccc cgcattcggg accgcctctc attcgaatag
      841 cgaacatgaa gagatccacc tcgccggctc gatccagccg catggcgcgc ttctggtcgt
      901 cagcgaacat gatcatcgcg tcatccaggc cagcgccaac gccgcggaat ttctgaatct
      961 cggaagcgta ctcggcgttc cgctcgccga gatcgacggc gatctgttga tcaagatcct
     1021 gccgcatctc gatcccaccg ccgaaggcat gccggtcgcg gtgcgctgcc ggatcggcaa
     1081 tccctctacg gagtactgcg gtctgatgca tcggcctccg gaaggcgggc tgatcatcga
     1141 actcgaacgt gccggcccgt cgatcgatct gtcaggcacg ctggcgccgg cgctggagcg
     1201 gatccgcacg gcgggttcac tgcgcgcgct gtgcgatgac accgtgctgc tgtttcagca
     1261 gtgcaccggc tacgaccggg tgatggtgta tcgtttcgat gagcaaggcc acggcctggt
     1321 attctccgag tgccatgtgc ctgggctcga atcctatttc ggcaaccgct atccgtcgtc
     1381 gactgtcccg cagatggcgc ggcagctgta cgtgcggcag cgcgtccgcg tgctggtcga
     1441 cgtcacctat cagccggtgc cgctggagcc gcggctgtcg ccgctgaccg ggcgcgatct
     1501 cgacatgtcg ggctgcttcc tgcgctcgat gtcgccgtgc catctgcagt tcctgaagga
     1561 catgggcgtg cgcgccaccc tggcggtgtc gctggtggtc ggcggcaagc tgtggggcct
     1621 ggttgtctgt caccattatc tgccgcgctt catccgtttc gagctgcggg cgatctgcaa
     1681 acggctcgcc gaaaggatcg cgacgcggat caccgcgctt gagagcggca tggacgagct
     1741 gtacaaggga gtctaacccg gtataacttc gtatagcata cattatacga agttattgtg
     1801 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     1861 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     1921 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     1981 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     2041 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt
     2101 ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc
     2161 tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     2221 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     2281 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     2341 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct
     2401 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     2461 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     2521 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     2581 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     2641 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     2701 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     2761 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     2821 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     2881 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     2941 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     3001 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     3061 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     3121 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     3181 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     3241 cattctagtt gtggtttgtc caaactcatc aatgtatctt attagataac ttcgtatagc
     3301 atacattata cgaagttatc tcgagtgtta acggcggccg ggatatcatg cctcgactgt
     3361 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     3421 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     3481 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     3541 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     3601 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     3661 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     3721 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3781 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     3841 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     3901 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     3961 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     4021 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     4081 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     4141 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     4201 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     4261 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     4321 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     4381 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa
     4441 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     4501 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     4561 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     4621 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg
     4681 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     4741 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     4801 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     4861 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     4921 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     4981 aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     5041 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     5101 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     5161 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     5221 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     5281 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     5341 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     5401 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     5461 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     5521 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     5581 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     5641 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     5701 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     5761 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     5821 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     5881 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     5941 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     6001 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     6061 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     6121 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     6181 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     6241 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     6301 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     6361 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     6421 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     6481 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     6541 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     6601 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     6661 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     6721 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     6781 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     6841 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     6901 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     6961 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     7021 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     7081 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     7141 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     7201 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     7261 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     7321 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     7381 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     7441 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     7501 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     7561 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     7621 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     7681 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     7741 gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct
     7801 cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
     7861 ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
     7921 caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
     7981 cagatatacg cgtt
//
