LOCUS       sequence-36637-e        5506 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5506)
  AUTHORS   Petersen LK, Stowers RS
  TITLE     A Gateway MultiSite Recombination Cloning Toolkit.
  JOURNAL   PLoS One. 2011;6(9):e24531. Epub 2011 Sep 9.
  PUBMED    21931740
REFERENCE   2  (bases 1 to 5506)
  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: "PLoS One.";
            date: "2011"; volume: "6(9)"; pages: "e24531. Epub 2011 Sep 9"
COMMENT     Sequence Label: pENTR L3-4XeGFP-Myc-L2
FEATURES             Location/Qualifiers
     source          1..5506
                     /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    570..665
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             join(689..691,692..1402)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(689..691,692..1402)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             1205..1930
                     /codon_start=1
                     /product="circularly permuted form of the Venus yellow 
                     fluorescent protein (Nagai et al., 2004)"
                     /label=cp173Venus
                     /note="efficient FRET acceptor for CFP"
                     /translation="DGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDH
                     MVLLEFVTAAGITLGMDELYKGGGSVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGE
                     GDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYV
                     QERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYI
                     MADKQKNGIKVNFKIRHNIE"
     CDS             join(1415..1417,1418..2128)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1415..1417,1418..2128)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             1931..2656
                     /codon_start=1
                     /product="circularly permuted form of the Venus yellow 
                     fluorescent protein (Nagai et al., 2004)"
                     /label=cp173Venus
                     /note="efficient FRET acceptor for CFP"
                     /translation="DGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDH
                     MVLLEFVTAAGITLGMDELYKGGGSVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGE
                     GDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYV
                     QERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYI
                     MADKQKNGIKVNFKIRHNIE"
     CDS             join(2141..2143,2144..2854)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(2141..2143,2144..2854)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             2657..3382
                     /codon_start=1
                     /product="circularly permuted form of the Venus yellow 
                     fluorescent protein (Nagai et al., 2004)"
                     /label=cp173Venus
                     /note="efficient FRET acceptor for CFP"
                     /translation="DGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDH
                     MVLLEFVTAAGITLGMDELYKGGGSVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGE
                     GDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYV
                     QERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYI
                     MADKQKNGIKVNFKIRHNIE"
     CDS             join(2867..2869,2870..3580)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(2867..2869,2870..3580)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     protein_bind    complement(3618..3717)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3747..3766)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3748..3766)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3771..3787)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3771..3787)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3889..3908)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3900..4709
                     /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(3967..3986)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4856..5444
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5345..5364
                     /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 tgtataataa
      661 agttggcggc aacggctccg gcaccggcgt gagcaagggc gaggagctgt tcaccggggt
      721 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
      781 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
      841 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
      901 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
      961 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     1021 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     1081 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     1141 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     1201 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     1261 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     1321 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     1381 cggcatggac gagctgtaca agggcggcgg ctccgtgagc aagggcgagg agctgttcac
     1441 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     1501 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     1561 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
     1621 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     1681 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     1741 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     1801 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     1861 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     1921 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     1981 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
     2041 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     2101 cactctcggc atggacgagc tgtacaaggg cggcggctcc gtgagcaagg gcgaggagct
     2161 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     2221 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     2281 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg
     2341 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     2401 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     2461 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     2521 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     2581 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
     2641 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     2701 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
     2761 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     2821 cgggatcact ctcggcatgg acgagctgta caagggcggc ggctccgtga gcaagggcga
     2881 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     2941 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
     3001 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac
     3061 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     3121 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa
     3181 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     3241 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta
     3301 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt
     3361 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa
     3421 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc
     3481 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     3541 cgccgccggg atcactctcg gcatggacga gctgtacaag ggcgacgagc agaagatctc
     3601 cgaggaggac ctgtaatacc cagctttctt gtacaaagtt ggcattataa gaaagcattg
     3661 cttatcaatt tgttgcaacg aacaggtcac tatcagtcaa aataaaatca ttatttgcca
     3721 tccagctgca gggcggccgc gatatcccct atagtgagtc gtattacatg gtcatagctg
     3781 tttcctggca gctctggccc gtgtctcaaa atctctgatg ttacattgca caagataaaa
     3841 atatatcatc atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta
     3901 tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt
     3961 tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgct
     4021 tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca
     4081 atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga
     4141 ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg
     4201 gaaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg
     4261 cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca
     4321 gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg
     4381 cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc
     4441 ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata
     4501 accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg
     4561 cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat
     4621 tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt
     4681 ttcatttgat gctcgatgag tttttctaat cagaattggt taattggttg taacactggc
     4741 agagcattac gctgacttga cgggacggcg caagctcatg accaaaatcc cttaacgtga
     4801 gttacgcgtc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     4861 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     4921 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     4981 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     5041 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     5101 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     5161 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     5221 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     5281 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     5341 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     5401 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     5461 cctttttacg gttcctggcc ttttgctggc cttttgctca catgtt
//
