LOCUS       sequence-165833-        4607 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  eGFP reporter donor containing double SA cutting sites for NHEJ 
            knock-in.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4607)
  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 4607)
  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: double cut NH-donor
FEATURES             Location/Qualifiers
     source          1..4607
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     623..642
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        623..641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     674..690
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     674..690
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_feature    734..1301
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    751..1303
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(895..912)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1122..1141
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(1315..1317,1318..1320,1321..2034)
                     /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(1315..1317,1318..1320,1321..2031)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1360..1381)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1621..1640)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1968..1989
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2066..2083)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2072..2296
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(2297..2330)
                     /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(2419..2439)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2419..2437)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2458..2474)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2458..2474)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2471..2493)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2482..2498
                     /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(2506..2512,2513..2530,2531..2536))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2551..2572
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2689..2706)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2860..3448)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2940..2959)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3619..4410,4411..4479))
                     /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     4242..4261
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4480..4584)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg agctccaccg
      661 cggtggcggc cgctctagaa ctagtggatc cgagatcgag tgccgcatca ccggcaattg
      721 acgcgtgctc ctctccctcc ccccccccta acgttactgg ccgaagccgc ttggaataag
      781 gccggtgtgc gtttgtctat atgttatttt ccaccatatt gccgtctttt ggcaatgtga
      841 gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg
      901 ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt
      961 gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca
     1021 ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc
     1081 agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat
     1141 tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc
     1201 ctcggtacac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga
     1261 accacgggga cgtggttttc ctttgaaaaa cacgatgata atatggccac aaccatggtg
     1321 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     1381 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     1441 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     1501 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
     1561 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     1621 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     1681 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     1741 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     1801 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     1861 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     1921 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     1981 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaaatgcat
     2041 cgatgatcta gagctcgctg atcagcctcg actgtgcctt ctagttgcca gctatctgtt
     2101 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     2161 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     2221 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     2281 gcggtgggct ctatggataa cttcgtatag catacattat acgaagttat cggaaccctt
     2341 aatgtcgacg agatcgagtg ccgcatcacc gggttaactc tctaacctcg agggggggcc
     2401 cggtaccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc
     2461 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     2521 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     2581 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     2641 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     2701 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     2761 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     2821 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     2881 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     2941 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     3001 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     3061 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     3121 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     3181 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     3241 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     3301 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     3361 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     3421 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     3481 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     3541 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     3601 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     3661 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     3721 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     3781 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     3841 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     3901 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     3961 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     4021 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     4081 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     4141 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     4201 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     4261 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     4321 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     4381 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     4441 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     4501 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     4561 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccac
//
