LOCUS       sequence-384218-        3129 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Selection plasmid expressing non-fluorescent EGFP A111V.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3129)
  AUTHORS   Vasquez CA, Evanoff M, Ranzau BL, Gu S, Deters E, Komor AC
  TITLE     Curing ""GFP-itis"" in Bacteria with Base Editors: Development of a 
            Genome Editing Science Program Implemented with High School Biology 
            Students.
  JOURNAL   CRISPR J. 2023 Apr 20. doi: 10.1089/crispr.2023.0002.
  PUBMED    37083425
REFERENCE   2  (bases 1 to 3129)
  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. 
            2023 Apr 20. doi: 10.1089/crispr.2023.0002."
COMMENT     Sequence Label: GFP-itis pSel
FEATURES             Location/Qualifiers
     source          1..3129
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     459..478
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     701..718
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      818..863
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             1032..1826
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1086..1105)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1696..1715
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      1853..1884
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     promoter        1918..1952
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     RBS             1961..1972
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(1979..1981,1982..1984,1985..2695)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRVEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2024..2045)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2285..2304)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2632..2653
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2708..2728
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2777..2794)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2947..3033
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      join(3108..3129,1..728)
                     /direction=RIGHT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
ORIGIN
        1 ttgatagacc gattgattca tcatctcata aataaagaaa aaccaccgct accaacggtg
       61 gttttctcaa ggttcgctga gctaccaact ctttgaacca aggtaagtgg gttggaggac
      121 cgcactcacc aaaatctgtt ctttcagttt agccttaaca ggtgcataac ttcaagacaa
      181 agtcctctaa atcagttacc aatggctgct gccagtggcg ataagtcgtg tcttaccggg
      241 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
      301 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacca acagcgtgag
      361 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
      421 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
      481 agtcctgtcg ggtttcgcca cctctggctt gagcgtcgat ttttgtgatg ctcgtcaggg
      541 gggcggagcc tatggaaaaa cgcctgcggc gttggcttct tccggtgctt tgctttttgc
      601 tcacatgttc tttccggctt tatcccctga ttctgtggat aaccgtatta ccgcttttga
      661 gtgagctgac accgctcgcc gcagtcgaac gaccgagcgt agcgagtcag tgagcgagga
      721 agcggaagtt ttctactgaa ccgctctaga tttcagtgca atttatctct tcaaatgtag
      781 cacctgaagt cagcccagga ggaagaggac atccggtcaa ataaaacgaa aggctcagtc
      841 gaaagactgg gcctttcgtt ttagacttag ggaccctaag ccaaataggc cgtccggaat
      901 tgccagctgg ggcgccctct ggtaaggttg ggaagccctg caaagtaaac tggatggctt
      961 tcttgccgcc aaggatctga tggcgcaggg gatcaagatc tgatcaagag acaggatgag
     1021 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     1081 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     1141 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     1201 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     1261 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     1321 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     1381 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     1441 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     1501 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     1561 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     1621 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     1681 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     1741 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     1801 atcgccttct tgacgagttc ttctaaactt aattaacggc actcctcagc caagtcaaaa
     1861 gcctccgacc ggaggctttt gactacatgc ccatggcgtt gtctgattcg ttaccaattg
     1921 acagctagct cagtcctagg tataatgcta gctactagtg aaagaggaga aatactagat
     1981 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     2041 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
     2101 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     2161 cgtgaccacc ctgacctatg gagtgcagtg cttcagccgc taccccgacc acatgaagca
     2221 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     2281 caaggacgac ggcaactaca agacccgcgt agaggtgaag ttcgagggcg acaccctggt
     2341 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     2401 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     2461 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     2521 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     2581 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     2641 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtctgg
     2701 tggttctccc aagaagaaaa ggaaagtcta agaattcgaa gcttggctgt tttggcggat
     2761 gagagaagat tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac
     2821 agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag
     2881 tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc
     2941 aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt
     3001 ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt gaaaagccaa
     3061 ataggccgtc actcggtcgc tacgctccgg gcgtgagact gcggcggaac ggaatagctg
     3121 ttcgttgac
//
