LOCUS       sequence-266841-        2874 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor-eGFP vector to be used in plasmid to plasmid integration 
            reporter assay. eGFP is conditionally expressed when integrated at 
            iCas9-sites on acceptor plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2874)
  AUTHORS   Standage-Beier K, Brookhouser N, Balachandran P, Zhang Q, Brafman 
            DA, Wang X
  TITLE     RNA-Guided Recombinase-Cas9 Fusion Targets Genomic DNA Deletion and 
            Integration.
  JOURNAL   CRISPR J. 2019 Aug;2:209-222. doi: 10.1089/crispr.2019.0013.
  PUBMED    31436506
REFERENCE   2  (bases 1 to 2874)
  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: "CRISPR J.";
            date: "2019-08"; volume: "2:209-222. doi"; pages: " 
            10.1089/crispr.2019.0013"
COMMENT     Sequence Label: pSB1C3-FeGFP
FEATURES             Location/Qualifiers
     source          1..2874
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      108..117
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(114..116,117..119,120..833)
                     /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(114..116,117..119,120..830)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(159..180)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(420..439)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     767..788
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     terminator      848..905
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     primer_bind     complement(929..946)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1100..1688)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1180..1199)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1871..1965)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(1986..2645)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2560..2579
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     2807..2825
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(2829..2872)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
ORIGIN
        1 gaattctccg atccttcccc caggcttgca ctcgtacgtt cgaaatatta taaattatca
       61 gacagacctt actccagaag ggggaaggct aggtggatcc accggtcgcc accatggtga
      121 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
      181 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
      241 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
      301 ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg
      361 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
      421 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
      481 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
      541 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca
      601 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
      661 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
      721 gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
      781 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag taaagcggcc
      841 gctgcagtcc ggcaaaaaag ggcaaggtgt caccaccctg ccctttttct ttaaaaccga
      901 aaagattact tcgcgttatg caggcttcct cgctcactga ctcgctgcgc tcggtcgttc
      961 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     1021 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     1081 aggccgcgtt gctggcgttt ttccacaggc tccgcccccc tgacgagcat cacaaaaatc
     1141 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     1201 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     1261 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     1321 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     1381 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     1441 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     1501 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     1561 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     1621 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     1681 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     1741 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     1801 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagct
     1861 cgaggcttgg attctcacca ataaaaaacg cccggcggca accgagcgtt ctgaacaaat
     1921 ccagatggag ttctgaggtc attactggat ctatcaacag gagtccaagc gagctcgata
     1981 tcaaattacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt aagcattctg
     2041 ccgacatgga agccatcaca aacggcatga tgaacctgaa tcgccagcgg catcagcacc
     2101 ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata
     2161 ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga gacgaaaaac
     2221 atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca cgccacatct
     2281 tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa
     2341 aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc ccatatcacc
     2401 agctcaccgt ctttcattgc catacgaaat tccggatgag cattcatcag gcgggcaaga
     2461 atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtctt taaaaaggcc
     2521 gtaatatcca gctgaacggt ctggttatag gtacattgag caactgactg aaatgcctca
     2581 aaatgttctt tacgatgcca ttgggatata tcaacggtgg tatatccagt gatttttttc
     2641 tccattttag cttccttagc tcctgaaaat ctcgataact caaaaaatac gcccggtagt
     2701 gatcttattt cattatggtg aaagttggaa cctcttacgt gcccgatcaa ctcgagtgcc
     2761 acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac
     2821 gaggcagaat ttcagataaa aaaaatcctt agctttcgct aaggatgatt tctg
//
