LOCUS       sequence-431464-        3328 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (Selection) fluorescent reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3328)
  AUTHORS   McGee AV, Liu YV, Griffith AL, Szegletes ZM, Wen B, Kraus C, Miller 
            NW, Steger RJ, Escude Velasco B, Bosch JA, Zirin JD, Viswanatha R, 
            Sontheimer EJ, Goodale A, Greene MA, Green TM, Doench JG
  TITLE     Modular vector assembly enables rapid assessment of emerging CRISPR 
            technologies.
  JOURNAL   Cell Genom. 2024 Mar 13;4(3):100519. doi: 
            10.1016/j.xgen.2024.100519.
  PUBMED    38484704
REFERENCE   2  (bases 1 to 3328)
  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: "Cell 
            Genom."; date: "2024-03-13"; volume: "4(3):100519. doi"; pages: " 
            10.1016/j.xgen.2024.100519"
COMMENT     Sequence Label: AA278
FEATURES             Location/Qualifiers
     source          1..3328
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     regulatory      444..453
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(450..452,453..455,456..1166)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(450..452,453..455,456..1166)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(495..516)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(756..775)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1103..1124
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1238..1254)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1238..1254)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1251..1273)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1262..1278
                     /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(1286..1292,1293..1310,1311..1316))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1331..1352
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1469..1486)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1640..2228)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1720..1739)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2406..3215)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3129..3148
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3207..3226
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagagaatt cgagctcggt acctcgcgaa
      421 tacatctaga tgaagacgta ggagccacca tggtgagcaa gggcgaggag ctgttcaccg
      481 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
      541 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
      601 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
      661 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
      721 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
      781 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
      841 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
      901 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
      961 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     1021 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     1081 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     1141 ctctcggcat ggacgagctg tacaagggaa gcacgtcttc atcggatccc gggcccgtcg
     1201 actgcagagg cctgcatgca agcttggtgt aatcatggtc atagctgttt cctgtgtgaa
     1261 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     1321 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     1381 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     1441 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     1501 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     1561 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     1621 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     1681 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     1741 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     1801 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     1861 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     1921 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     1981 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     2041 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     2101 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     2161 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     2221 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     2281 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     2341 attaaaaatg aagttttaaa tcaagcccaa tctgaataat gttacaacca attaaccaat
     2401 tctgattaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta
     2461 tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag
     2521 ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
     2581 caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg
     2641 acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca
     2701 ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt
     2761 gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga
     2821 atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca
     2881 ggatattctt ctaatacctg gaatgctgtt tttccgggga tcgcagtggt gagtaaccat
     2941 gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc
     3001 cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc
     3061 agaaacaact ctggcgcatc gggcttccca tacaagcgat agattgtcgc acctgattgc
     3121 ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat
     3181 cgcggcctcg acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg
     3241 taagcagaca gttttattgt tcatgatgat atatttttat cttgtgcaat gtaacatcag
     3301 agattttgag acacgggcca gagctgca
//
