LOCUS       sequence-314576-        6431 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 6431)
  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 6431)
  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
FEATURES             Location/Qualifiers
     source          1..6431
                     /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"
     primer_bind     complement(1788..1805)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1794..2018
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2064..2492
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2151..2170)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2361..2382
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2501..2521)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2506..2835
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2686..2821
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2748..2767
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2902..3696
                     /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(2956..2975)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3566..3585
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3870..3991
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3907..3926)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3961..3980
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4040..4056)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4040..4056)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4053..4075)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4064..4080
                     /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(4088..4094,4095..4112,4113..4118))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4133..4154
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4271..4288)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4442..5030)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4522..4541)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5201..5992,5993..6061))
                     /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     5824..5843
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6062..6166)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6241..6260)
                     /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 gtctaactcg agtgttaacg gcggccggga tatcatgcct cgactgtgcc
     1801 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     1861 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     1921 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     1981 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     2041 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     2101 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     2161 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     2221 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     2281 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     2341 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     2401 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     2461 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     2521 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     2581 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     2641 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     2701 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     2761 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     2821 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
     2881 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     2941 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     3001 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     3061 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     3121 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     3181 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     3241 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     3301 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     3361 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     3421 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     3481 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     3541 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     3601 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     3661 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     3721 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     3781 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     3841 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     3901 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     3961 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     4021 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     4081 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     4141 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4201 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     4261 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4321 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4381 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4441 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4501 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4561 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4621 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4681 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4741 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4801 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4861 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     4921 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4981 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5041 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5101 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     5161 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     5221 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     5281 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     5341 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     5401 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5461 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     5521 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     5581 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     5641 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     5701 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     5761 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     5821 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     5881 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     5941 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     6001 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     6061 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     6121 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     6181 aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag
     6241 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga
     6301 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa
     6361 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag
     6421 atatacgcgt t
//
