LOCUS       sequence-304795-        3866 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Middle entry vector containing QUAS5x element upstream of GFP-NLS..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3866)
  AUTHORS   Burgess J, Burrows JT, Sadhak R, Chiang S, Weiss A, D'Amata C, 
            Molinaro AM, Zhu S, Long M, Hu C, Krause HM, Pearson BJ
  TITLE     An optimized QF-binary expression system for use in zebrafish.
  JOURNAL   Dev Biol. 2020 Jul 19. pii: S0012-1606(20)30202-5. doi: 
            10.1016/j.ydbio.2020.07.007.
  PUBMED    32697972
REFERENCE   2  (bases 1 to 3866)
  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: "Dev Biol. 
            2020 Jul 19. pii: S0012-1606(20)30202-5. doi: 
            10.1016/j.ydbio.2020.07.007."
COMMENT     Sequence Label: pME-QUAS5x:GFPNLS-SV40pA
FEATURES             Location/Qualifiers
     source          1..3866
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      926..935
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(932..934,935..937,938..1648)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(932..934,935..937,938..1648)
                     /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(977..998)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1238..1257)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1585..1606
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1831..1850)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1885..1904
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1948..1964)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1949..1965)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     protein_bind    complement(1991..2090)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(2108..2126)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2131..2147)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2131..2147)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2249..2268)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2260..3069
                     /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     complement(2327..2346)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3216..3804
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3705..3724
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctcg aattggagct ccaccgcggt ggcggccgca aggataaaca attatcctca
      721 aggataaaca attatcctca aggataaaca attatcctca aggataaaca attatcctca
      781 aggataaaca attatcctcg caagggtcga ctctagaggg tatataatgg atcccatcgc
      841 gtctcagcct cactttgagc tcctccacac gaattccctc gacctcgaag acgcgtctgt
      901 ttgggatcca ccggtcacta gtgccgccac catggtgagc aagggcgagg agctgttcac
      961 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     1021 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     1081 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
     1141 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     1201 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     1261 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     1321 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     1381 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     1441 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     1501 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
     1561 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     1621 cactctcggc atggacgagc tgtacaagag caggcacaga aggcatcgcc agcgctctag
     1681 gagccgcaat cgcagccgaa gtcgcagcag tgaacgaaaa cgccgtcaac ggagccgaag
     1741 tcgcagcagt gaacgaagac gctacttgta caagtaatct agaactatag tgagtcgtat
     1801 tacgtagatc cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg
     1861 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     1921 ataagctgca ataaacaaga agcttatcga taccgtcgac ctcgaggggg ggcccggtac
     1981 ccagcttttg acccagcttt cttgtacaaa gttggcatta taagaaagca ttgcttatca
     2041 atttgttgca acgaacaggt cactatcagt caaaataaaa tcattatttg ccatccagct
     2101 gatatcccct atagtgagtc gtattacatg gtcatagctg tttcctggca gctctggccc
     2161 gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata
     2221 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa
     2281 acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct
     2341 cgcgataatg tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg
     2401 ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg
     2461 gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt
     2521 actcctgatg atgcatggtt actcaccact gcgatccccg gaaaaacagc attccaggta
     2581 ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc
     2641 cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc
     2701 gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag
     2761 cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca
     2821 ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg
     2881 aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt
     2941 gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa
     3001 aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag
     3061 tttttctaat cagaattggt taattggttg taacactggc agagcattac gctgacttga
     3121 cgggacggcg caagctcatg accaaaatcc cttaacgtga gttacgcgtc gttccactga
     3181 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     3241 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     3301 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     3361 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     3421 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     3481 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     3541 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     3601 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     3661 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     3721 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     3781 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     3841 ttttgctggc cttttgctca catgtt
//
