LOCUS       sequence-179972-        6474 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express eGFP reporter for HDR-induced knock-in.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6474)
  AUTHORS   He X, Tan C, Wang F, Wang Y, Zhou R, Cui D, You W, Zhao H, Ren J, 
            Feng B
  TITLE     Knock-in of large reporter genes in human cells via 
            CRISPR/Cas9-induced homology-dependent and independent DNA repair.
  JOURNAL   Nucleic Acids Res. 2016 May 19;44(9):e85. doi: 10.1093/nar/gkw064. 
            Epub 2016 Feb 4.
  PUBMED    26850641
REFERENCE   2  (bases 1 to 6474)
  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: "Nucleic 
            Acids Res."; date: "2016-05-19"; volume: "44(9):e85. doi"; pages: " 
            10.1093/nar/gkw064. Epub 2016 Feb 4"
COMMENT     Sequence Label: ires-eGFP(+HAs) donor-1
FEATURES             Location/Qualifiers
     source          1..6474
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     27..49
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     41..58
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     42..58
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     65..84
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        65..83
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     116..132
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     116..132
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_feature    1099..1666
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    1116..1668
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1260..1277)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1487..1506
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(1680..1682,1683..1685,1686..2399)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1680..1682,1683..1685,1686..2396)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1725..1746)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1986..2005)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2333..2354
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2431..2448)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2437..2661
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(2662..2695)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        complement(3728..3746)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3767..3783)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3767..3783)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3780..3802)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3791..3807
                     /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(3815..3821,3822..3839,3840..3845))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3860..3881
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3998..4015)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4169..4757)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4249..4268)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4928..5719,5720..5788))
                     /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     5551..5570
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5789..5893)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5919..6374)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6056..6077)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6268..6287
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga
       61 attgtaatac gactcactat agggcgaatt ggagctccac cgcggtggcg gccgctctag
      121 aactagtgga tccgacacgc tcccctgact tgcgccccgc tccctctttc tttgcagcaa
      181 tgcctcctgc accaccaact gcttagcacc cctggccaag gtcatccatg acaactttgg
      241 tatcgtggaa ggactcatgg tatgagagct ggggaatggg actgaggctc ccacctttct
      301 catccaagac tggctcctcc ctgccggggc tgcgtgcaac cctggggttg ggggttctgg
      361 ggactggctt tcccataatt tcctttcaag gtggggaggg aggtagaggg gtgatgtggg
      421 gagtacgctg cagggcctca ctccttttgc agaccacagt ccatgccatc actgccaccc
      481 agaagactgt ggatggcccc tccgggaaac tgtggcgtga tggccgcggg gctctccaga
      541 acatcatccc tgcctctact ggcgctgcca aggctgtggg caaggtcatc cctgagctga
      601 acgggaagct cactggcatg gccttccgtg tccccactgc caacgtgtca gtggtggacc
      661 tgacctgccg tctagaaaaa cctgccaaat atgatgacat caagaaggtg gtgaagcagg
      721 cgtcggaggg ccccctcaag ggcatcctgg gctacactga gcaccaggtg gtctcctctg
      781 acttcaacag cgacacccac tcctccacct tcgacgctgg ggctggcatt gccctcaacg
      841 accactttgt caagctcatt tcctggtatg tggctggggc cagagactgg ctcttaaaaa
      901 gtgcagggtc tggcgccctc tggtggctgg ctcagaaaaa gggccctgac aactcttttc
      961 atcttctagg tatgacaacg aatttggcta cagcaacagg gtggtggacc tcatggccca
     1021 catggcctcc aaggagtaag acccctggac caccagcccc agcaagagca caagaggaac
     1081 aattgacgcg tgctcctctc cctccccccc ccntaacgtt actggccgaa gccgcttgga
     1141 ataaggccgg tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa
     1201 tgtgagggcc cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc
     1261 tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc
     1321 ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg
     1381 cgacaggtgc ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca
     1441 accccagtgc cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag
     1501 cgtattcaac aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct
     1561 ggggcctcgg tacacatgct ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc
     1621 cccgaaccac ggggacgtgg ttttcctttg aaaaacacga tgataatatg gccacaacca
     1681 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     1741 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     1801 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     1861 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     1921 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     1981 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     2041 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     2101 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     2161 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     2221 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     2281 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     2341 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaaa
     2401 tgcatcgatg atctagagct cgctgatcag cctcgactgt gccttctagt tgccagctat
     2461 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     2521 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     2581 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     2641 gggatgcggt gggctctatg gataacttcg tatagcatac attatacgaa gttatcggaa
     2701 cccttaatgt cgacccctgc cacactcagt cccccaccac actgaatctc ccctcctcac
     2761 agttgccatg tagacccctt gaagagggga ggggcctagg gagccgcacc ttgtcatgta
     2821 ccatcaataa agtaccctgt gctcaaccag ttacttgtcc tgtcttattc tagggtctgg
     2881 ggcagagggg agggaagctg ggcttgtgtc aaggtgagac attcttgctg gggagggacc
     2941 tggtatgttc tcctcagact gagggtaggg cctccaaaca gccttgcttg cttcgagaac
     3001 catttgcttc ccgctcagac gtcttgagtg ctacaggaag ctggcaccac tacttcagag
     3061 aacaaggcct tttcctctcc tcgctccagt cctaggctat ctgctgttgg ccaaacatgg
     3121 aagaagctat tctgtgggcg gccccaggga ggctgacagg tggaggaagt cagggctcgc
     3181 actgggctct gacgctgact ggttagtgga gctcagcctg gagctgagct gcagcgggca
     3241 attccagctt ggcctccgca gctgtgaggt cttgagcacg tgctctattg ctttctgtgc
     3301 cctcgtgtct tatctgagga catcgtggcc agcccctaag gtcttcaagc aggattcatc
     3361 taggtaaacc aagtacctaa aaccatgccc aaggcggtaa ggactatata atgtttaaaa
     3421 atcggtaaaa atgcccacct cgcatagttt tgaggaagat gaactgagat gtgtcagggt
     3481 gacttatttc catcatcgtc cttaggggag cttgggtagg ggcaaggcgt gtagctggga
     3541 cctaggtcca gacccctggc tctgccactg aacggctcag gtgctttggg cagttactcc
     3601 ccggcctcac tttgcacgtg tgcttaccta gtggagacaa aagtacatac ctcggtagag
     3661 cgcgcacgcc tgtaacccca ggttaactct ctaacctcga gggggggccc ggtaccagct
     3721 tttgttccct ttagtgaggg ttaatttcga gcttggcgta atcatggtca tagctgtttc
     3781 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     3841 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     3901 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     3961 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     4021 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     4081 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     4141 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     4201 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     4261 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4321 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4381 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4441 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4501 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4561 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     4621 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4681 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4741 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4801 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4861 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     4921 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     4981 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     5041 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     5101 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     5161 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     5221 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     5281 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5341 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5401 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5461 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     5521 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     5581 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     5641 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     5701 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     5761 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     5821 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     5881 taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt
     5941 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     6001 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     6061 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     6121 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     6181 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     6241 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     6301 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     6361 gctacagggc gcgtcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt
     6421 gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcg
//
