LOCUS       sequence-314572-        7988 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 7988)
  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 7988)
  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-LoxP-Neo-N-miRFP670-C
FEATURES             Location/Qualifiers
     source          1..7988
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..329
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      180..315
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             396..1190
                     /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    1364..1485
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(1490..1523)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1563..2507
                     /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(2569..2586)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2575..2799
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2845..3273
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2932..2951)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3142..3163
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3287..3616
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3467..3602
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3683..4477
                     /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    4651..4772
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4821..4837)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4821..4837)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4834..4856)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4845..4861
                     /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(4869..4875,4876..4893,4894..4899))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4914..4935
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5052..5069)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5223..5811)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5303..5322)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5982..6773,6774..6842))
                     /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     6605..6624
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6843..6947)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7022..7041)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7213..7592
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7593..7796
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7747..7767
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     7841..7860
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7841..7859
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(7955..7988)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
       61 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
      121 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
      181 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
      241 ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag
      301 gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg
      361 gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg
      421 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
      481 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
      541 tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt
      601 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
      661 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
      721 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
      781 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
      841 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
      901 aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg
      961 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact
     1021 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg
     1081 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
     1141 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct
     1201 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac
     1261 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     1321 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc
     1381 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     1441 actgcattct agttgtggtt tgtccaaact catcaatgta tcttattaga taacttcgta
     1501 tagcatacat tatacgaagt tattctagca tcgaattccc tagaggcggc ggcggcgtcg
     1561 agatggtagc aggtcatgcc tctggcagcc ccgcattcgg gaccgcctct cattcgaata
     1621 gcgaacatga agagatccac ctcgccggct cgatccagcc gcatggcgcg cttctggtcg
     1681 tcagcgaaca tgatcatcgc gtcatccagg ccagcgccaa cgccgcggaa tttctgaatc
     1741 tcggaagcgt actcggcgtt ccgctcgccg agatcgacgg cgatctgttg atcaagatcc
     1801 tgccgcatct cgatcccacc gccgaaggca tgccggtcgc ggtgcgctgc cggatcggca
     1861 atccctctac ggagtactgc ggtctgatgc atcggcctcc ggaaggcggg ctgatcatcg
     1921 aactcgaacg tgccggcccg tcgatcgatc tgtcaggcac gctggcgccg gcgctggagc
     1981 ggatccgcac ggcgggttca ctgcgcgcgc tgtgcgatga caccgtgctg ctgtttcagc
     2041 agtgcaccgg ctacgaccgg gtgatggtgt atcgtttcga tgagcaaggc cacggcctgg
     2101 tattctccga gtgccatgtg cctgggctcg aatcctattt cggcaaccgc tatccgtcgt
     2161 cgactgtccc gcagatggcg cggcagctgt acgtgcggca gcgcgtccgc gtgctggtcg
     2221 acgtcaccta tcagccggtg ccgctggagc cgcggctgtc gccgctgacc gggcgcgatc
     2281 tcgacatgtc gggctgcttc ctgcgctcga tgtcgccgtg ccatctgcag ttcctgaagg
     2341 acatgggcgt gcgcgccacc ctggcggtgt cgctggtggt cggcggcaag ctgtggggcc
     2401 tggttgtctg tcaccattat ctgccgcgct tcatccgttt cgagctgcgg gcgatctgca
     2461 aacggctcgc cgaaaggatc gcgacgcgga tcaccgcgct tgagagcggc atggacgagc
     2521 tgtacaaggg agtctaactc gagtgttaac ggcggccggg atatcatgcc tcgactgtgc
     2581 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     2641 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     2701 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     2761 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
     2821 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     2881 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     2941 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     3001 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     3061 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     3121 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     3181 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     3241 atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg
     3301 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     3361 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     3421 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     3481 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     3541 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     3601 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga
     3661 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     3721 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     3781 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     3841 gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac
     3901 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     3961 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     4021 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     4081 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     4141 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     4201 gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     4261 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     4321 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     4381 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     4441 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     4501 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     4561 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     4621 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     4681 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     4741 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     4801 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     4861 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     4921 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     4981 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     5041 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     5101 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     5161 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     5221 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     5281 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     5341 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     5401 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     5461 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     5521 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     5581 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     5641 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     5701 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     5761 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     5821 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     5881 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     5941 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     6001 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     6061 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     6121 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     6181 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     6241 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     6301 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     6361 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     6421 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     6481 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     6541 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     6601 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     6661 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     6721 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     6781 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     6841 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     6901 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     6961 aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca
     7021 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg
     7081 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
     7141 attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca
     7201 gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
     7261 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
     7321 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
     7381 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
     7441 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
     7501 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
     7561 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
     7621 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
     7681 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
     7741 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctct
     7801 ggctaactag agaacccact gcttactggc ttatcgaaat taatacgact cactataggg
     7861 agacccaagc ttggtaccga gctcggatcc actagtaacg gccgccagtg tgctggaatt
     7921 ctgctcgggc ccttctagac aatcgatcac cggtataact tcgtatagca tacattatac
     7981 gaagttat
//
