LOCUS       sequence-431397-        3409 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (2A Selection) violet-excited GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3409)
  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 3409)
  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: AA050
FEATURES             Location/Qualifiers
     source          1..3409
                     /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"
     CDS             444..500
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(513..515,516..1229)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLFIQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(555..576)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(816..835)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1163..1184
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1319..1335)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1319..1335)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1332..1354)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1343..1359
                     /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(1367..1373,1374..1391,1392..1397))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1412..1433
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1550..1567)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1721..2309)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1801..1820)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2487..3296)
                     /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     3210..3229
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3288..3307
                     /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 tgaagacgga agcgcaacaa acttctctct gctgaaacaa gccggagatg
      481 tcgaagagaa tcctggaccg gcaccgggat ccgtgagcaa gggcgaggag ctgttcaccg
      541 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
      601 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
      661 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cttcagctac ggcgtgcagt
      721 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
      781 aaggctacgt ccaggagcgc accatcagct tcaaggacga cggcaactac aagacccgcg
      841 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
      901 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
      961 tctatatcac ggccgacaag cagaagaacg gcatcaaggc gaacttcaag atccgccaca
     1021 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     1081 acggccccgt gctgctgccc gacaaccact acctgttcat ccagtccgcc ctgagcaaag
     1141 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     1201 ctcacggcat ggacgagctg tacaagtaag gatcctgata aattccagtc ttcggaacca
     1261 aatcggatcc cgggcccgtc gactgcagag gcctgcatgc aagcttggtg taatcatggt
     1321 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     1381 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     1441 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     1501 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     1561 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     1621 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     1681 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     1741 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     1801 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     1861 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     1921 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     1981 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     2041 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     2101 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     2161 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     2221 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     2281 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     2341 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     2401 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaagccca atctgaataa
     2461 tgttacaacc aattaaccaa ttctgattag aaaaactcat cgagcatcaa atgaaactgc
     2521 aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa
     2581 ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt
     2641 ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca
     2701 agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt
     2761 tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca
     2821 accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta
     2881 aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca
     2941 acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt ttttccgggg
     3001 atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga
     3061 agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca
     3121 acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaagcga
     3181 tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca
     3241 gcatccatgt tggaatttaa tcgcggcctc gacgtttccc gttgaatatg gctcataaca
     3301 ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgatga tatattttta
     3361 tcttgtgcaa tgtaacatca gagattttga gacacgggcc agagctgca
//
