LOCUS       sequence-420595-        7600 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway cloning compatible vector for transient expression in 
            protoplasts. Arabidopsis UBQ10 promoter expression of N-terminal YFP
            fusion protein. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7600)
  AUTHORS   Allen JR, Flanagan M, Pathak S, Emenecker SK, Emenecker RJ, Strader 
            LC
  TITLE     An improved toolkit of gateway- and gibson assembly-compatible 
            vectors for protoplast transfection and agrobacterium-mediated plant
            transformation.
  JOURNAL   BMC Res Notes. 2025 Apr 8;18(1):149. doi: 
            10.1186/s13104-025-07231-1.
  PUBMED    40200324
REFERENCE   2  (bases 1 to 7600)
  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: "BMC Res 
            Notes."; date: "2025-04-8"; volume: "18(1):149. doi"; pages: " 
            10.1186/s13104-025-07231-1"
COMMENT     Sequence Label: pLCS100
FEATURES             Location/Qualifiers
     source          1..7600
                     /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"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    2010..2064
                     /label=TMV Omega
                     /note="translational enhancer from the tobacco mosaic virus
                     5'-leader sequence (Gallie et al., 1988)"
     CDS             join(2066..2068,2069..2071,2072..2782)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2111..2132)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2372..2391)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2719..2740
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    2852..2976
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(3001..3006,3007..3024,3025..3031)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             3085..3744
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3151..3170)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             4086..4391
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     4270..4289
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(4432..4556)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     terminator      4615..5322
                     /label=OCS terminator
                     /note="octopine synthase terminator"
     primer_bind     complement(5379..5395)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5379..5395)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5392..5414)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5403..5419
                     /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(5427..5433,5434..5451,5452..5457))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5472..5493
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5610..5627)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5781..6369)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5861..5880)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6540..7331,7332..7400))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7163..7182
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7401..7505)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7573..7591
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     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 ccagtgccaa gctttcgcga agcacccacc
      421 ctaagccaca ttttaatcct tctgttggcc atgccccatc aaagttgcac ttaacccaag
      481 attgtggtgg agcttcccat gtttctcgtc tgtcccgacg gtgttgtggt tggtgctttc
      541 cttacattct gagcctcttt ccttctaatc cactcatctg catcttcttg tgtccttact
      601 aatacctcat tggttccaaa ttccctccct ttaagcacca gctcgtttct gttcttccac
      661 agcctcccaa gtatccaagg gactaaagcc tccacattct tcagatcagg atattcttgt
      721 ttaagatgtt gaactctatg gaggtttgta tgaactgatg atctaggacc ggataagttc
      781 ccttcttcat agcgaactta ttcaaagaat gttttgtgta tcattcttgt tacattgtta
      841 ttaatgaaaa aatattattg gtcattggac tgaacacgag tgttaaatat ggaccaggcc
      901 ccaaataaga tccattgata tatgaattaa ataacaagaa taaatcgagt caccaaacca
      961 cttgcctttt ttaacgagac ttgttcacca acttgataca aaagtcatta tcctatgcaa
     1021 atcaataatc atacaaaaat atccaataac actaaaaaat taaaagaaat ggataatttc
     1081 acaatatgtt atacgataaa gaagttactt ttccaagaaa ttcactgatt ttataagccc
     1141 acttgcatta gataaatggc aaaaaaaaac aaaaaggaaa agaaataaag cacgaagaat
     1201 tctagaaaat acgaaatacg cttcaatgca gtgggaccca cggttcaatt attgccaatt
     1261 ttcagctcca ccgtatattt aaaaaataaa acgataatgc taaaaaaata taaatcgtaa
     1321 cgatcgttaa atctcaacgg ctggatctta tgacgaccgt tagaaattgt ggttgtcgac
     1381 gagtcagtaa taaacggcgt caaagtggtt gcagccggca cacacgagtc gtgtttatca
     1441 actcaaagca caaatacttt tcctcaacct aaaaataagg caattagcca aaaacaactt
     1501 tgcgtgtaaa caacgctcaa tacacgtgtc attttattat tagctattgc ttcaccgcct
     1561 tagctttctc gtgacctagt cgtcctcgtc ttttcttctt cttcttctat aaaacaatac
     1621 ccaaagagct cttcttcttc acaattcaga tttcaatttc tcaaaatctt aaaaactttc
     1681 tctcaattct ctctaccgtg atcaaggtaa atttctgtgt tccttattct ctcaaaatct
     1741 tcgattttgt tttcgttcga tcccaatttc gtatatgttc tttggtttag attctgttaa
     1801 tcttagatcg aagacgattt tctgggtttg atcgttagat atcatcttaa ttctcgatta
     1861 gggtttcata gatatcatcc gatttgttca aataatttga gttttgtcga ataattactc
     1921 ttcgatttgt gatttctatc tagatctggt gttagtttct agtttgtgcg atcgaatttg
     1981 tcctcgagta taagagctct atttttacaa caattaccaa caacaacaaa caacaaacaa
     2041 cattacaatt acatttacaa ttaccatggt gagcaagggc gaggagctgt tcaccggggt
     2101 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     2161 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     2221 caagctgccc gtgccctggc ccaccctcgt gaccaccttc ggctacggcc tgcagtgctt
     2281 cgcccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     2341 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     2401 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     2461 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     2521 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     2581 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     2641 ccccgtgctg ctgcccgaca accactacct gagctaccag tccgccctga gcaaagaccc
     2701 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     2761 cggcatggac gagctgtaca agtccggact cagatcacga gctcaagctt caaattctgc
     2821 agtcgacggt accgcgggcc cgggatcatc aacaagtttg tacaaaaaag ctgaacgaga
     2881 aacgtaaaat gatataaata tcaatatatt aaattagatt ttgcataaaa aacagactac
     2941 ataatactgt aaaacacaac atatccagtc actatggcgg ccgcattagg caccccaggc
     3001 tttacacttt atgcttccgg ctcgtataat gtgtggattt tgagttagga tccgtcgaga
     3061 ttttcaggag ctaaggaagc taaaatggag aaaaaaatca ctggatatac caccgttgat
     3121 atatcccaat ggcatcgtaa agaacatttt gaggcatttc agtcagttgc tcaatgtacc
     3181 tataaccaga ccgttcagct ggatattacg gcctttttaa agaccgtaaa gaaaaataag
     3241 cacaagtttt atccggcctt tattcacatt cttgcccgcc tgatgaatgc tcatccggag
     3301 ttccgtatgg caatgaaaga cggtgagctg gtgatatggg atagtgttca cccttgttac
     3361 accgttttcc atgagcaaac tgaaacgttt tcatcgctct ggagtgaata ccacgacgat
     3421 ttccggcagt ttctacacat atattcgcaa gatgtggcgt gttacggtga aaacctggcc
     3481 tatttcccta aagggtttat tgagaatatg tttttcgtct cagccaatcc ctgggtgagt
     3541 ttcaccagtt ttgatttaaa cgtggccaat atggacaact tcttcgcccc cgttttcacc
     3601 atgggcaaat attatacgca aggcgacaag gtgctgatgc cgctggcgat tcaggttcat
     3661 catgccgttt gtgatggctt ccatgtcggc agaatgctta atgaattaca acagtactgc
     3721 gatgagtggc agggcggggc gtaaagatct ggatccggct tactaaaagc cagataacag
     3781 tatgcgtatt tgcgcgctga tttttgcggt ataagaatat atactgatat gtatacccga
     3841 agtatgtcaa aaagaggtat gctatgaagc agcgtattac agtgacagtt gacagcgaca
     3901 gctatcagtt gctcaaggca tatatgatgt caatatctcc ggtctggtaa gcacaaccat
     3961 gcagaatgaa gcccgtcgtc tgcgtgccga acgctggaaa gcggaaaatc aggaagggat
     4021 ggctgaggtc gcccggttta ttgaaatgaa cggctctttt gctgacgaga acaggggctg
     4081 gtgaaatgca gtttaaggtt tacacctata aaagagagag ccgttatcgt ctgtttgtgg
     4141 atgtacagag tgatattatt gacacgcccg ggcgacggat ggtgatcccc ctggccagtg
     4201 cacgtctgct gtcagataaa gtctcccgtg aactttaccc ggtggtgcat atcggggatg
     4261 aaagctggcg catgatgacc accgatatgg ccagtgtgcc ggtctccgtt atcggggaag
     4321 aagtggctga tctcagccac cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct
     4381 ggggaatata aatgtcaggc tcccttatac acagccagtc tgcaggtcga ccatagtgac
     4441 tggatatgtt gtgttttaca gtattatgta gtctgttttt tatgcaaaat ctaatttaat
     4501 atattgatat ttatatcatt ttacgtttct cgttcagctt tcttgtacaa agtggttgat
     4561 gatcctaggt gagtctagag agttaattaa gacccgggac tagtccctag agtcctgctt
     4621 taatgagata tgcgagacgc ctatgatcgc atgatatttg ctttcaattc tgttgtgcac
     4681 gttgtaaaaa acctgagcat gtgtagctca gatccttacc gccggtttcg gttcattcta
     4741 atgaatatat cacccgttac tatcgtattt ttatgaataa tattctccgt tcaatttact
     4801 gattgtaccc tactacttat atgtacaata ttaaaatgaa aacaatatat tgtgctgaat
     4861 aggtttatag cgacatctat gatagagcgc cacaataaca aacaattgcg ttttattatt
     4921 acaaatccaa ttttaaaaaa agcggcagaa ccggtcaaac ctaaaagact gattacataa
     4981 atcttattca aatttcaaaa gtgccccagg ggctagtatc tacgacacac cgagcggcga
     5041 actaataacg ctcactgaag ggaactccgg ttccccgccg gcgcgcatgg gtgagattcc
     5101 ttgaagttga gtattggccg tccgctctac cgaaagttac gggcaccatt caacccggtc
     5161 cagcacggcg gccgggtaac cgacttgctg ccccgagaat tatgcagcat ttttttggtg
     5221 tatgtgggcc ccaaatgaag tgcaggtcaa accttgacag tgacgacaaa tcgttgggcg
     5281 ggtccagggc gaattttgcg acaacatgtc gaggctcagc aggacctgca ggtcgactct
     5341 agaggatccc cgggtaccga gctcgaattc gtaatcatgt catagctgtt tcctgtgtga
     5401 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     5461 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     5521 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     5581 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     5641 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     5701 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     5761 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     5821 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5881 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5941 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     6001 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     6061 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     6121 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     6181 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     6241 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     6301 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     6361 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     6421 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     6481 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     6541 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     6601 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     6661 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     6721 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     6781 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     6841 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     6901 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     6961 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     7021 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     7081 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     7141 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     7201 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     7261 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     7321 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     7381 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     7441 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     7501 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     7561 ttaacctata aaaataggcg tatcacgagg ccctttcgtc
//
