LOCUS       sequence-420435-        6806 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway cloning compatible vector for transient expression in 
            protoplasts. Arabidopsis UBQ10 promoter expression of untagged 
            protein. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6806)
  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 6806)
  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: pLCS99
FEATURES             Location/Qualifiers
     source          1..6806
                     /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"
     protein_bind    2058..2182
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(2207..2212,2213..2230,2231..2237)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2291..2950
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2357..2376)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             3292..3597
                     /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     3476..3495
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(3638..3762)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     terminator      3821..4528
                     /label=OCS terminator
                     /note="octopine synthase terminator"
     primer_bind     complement(4585..4601)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4585..4601)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4598..4620)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4609..4625
                     /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(4633..4639,4640..4657,4658..4663))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4678..4699
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4816..4833)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4987..5575)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5067..5086)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5746..6537,6538..6606))
                     /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     6369..6388
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6607..6711)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6779..6797
                     /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 actttcgcga 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 tcctcgagtc cggactcaga tcacgagctc aagcttcaaa ttctgcagtc gacggtaccg
     2041 cgggcccggg atcatcaaca agtttgtaca aaaaagctga acgagaaacg taaaatgata
     2101 taaatatcaa tatattaaat tagattttgc ataaaaaaca gactacataa tactgtaaaa
     2161 cacaacatat ccagtcacta tggcggccgc attaggcacc ccaggcttta cactttatgc
     2221 ttccggctcg tataatgtgt ggattttgag ttaggatccg tcgagatttt caggagctaa
     2281 ggaagctaaa atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca
     2341 tcgtaaagaa cattttgagg catttcagtc agttgctcaa tgtacctata accagaccgt
     2401 tcagctggat attacggcct ttttaaagac cgtaaagaaa aataagcaca agttttatcc
     2461 ggcctttatt cacattcttg cccgcctgat gaatgctcat ccggagttcc gtatggcaat
     2521 gaaagacggt gagctggtga tatgggatag tgttcaccct tgttacaccg ttttccatga
     2581 gcaaactgaa acgttttcat cgctctggag tgaataccac gacgatttcc ggcagtttct
     2641 acacatatat tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg
     2701 gtttattgag aatatgtttt tcgtctcagc caatccctgg gtgagtttca ccagttttga
     2761 tttaaacgtg gccaatatgg acaacttctt cgcccccgtt ttcaccatgg gcaaatatta
     2821 tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtttgtga
     2881 tggcttccat gtcggcagaa tgcttaatga attacaacag tactgcgatg agtggcaggg
     2941 cggggcgtaa agatctggat ccggcttact aaaagccaga taacagtatg cgtatttgcg
     3001 cgctgatttt tgcggtataa gaatatatac tgatatgtat acccgaagta tgtcaaaaag
     3061 aggtatgcta tgaagcagcg tattacagtg acagttgaca gcgacagcta tcagttgctc
     3121 aaggcatata tgatgtcaat atctccggtc tggtaagcac aaccatgcag aatgaagccc
     3181 gtcgtctgcg tgccgaacgc tggaaagcgg aaaatcagga agggatggct gaggtcgccc
     3241 ggtttattga aatgaacggc tcttttgctg acgagaacag gggctggtga aatgcagttt
     3301 aaggtttaca cctataaaag agagagccgt tatcgtctgt ttgtggatgt acagagtgat
     3361 attattgaca cgcccgggcg acggatggtg atccccctgg ccagtgcacg tctgctgtca
     3421 gataaagtct cccgtgaact ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg
     3481 atgaccaccg atatggccag tgtgccggtc tccgttatcg gggaagaagt ggctgatctc
     3541 agccaccgcg aaaatgacat caaaaacgcc attaacctga tgttctgggg aatataaatg
     3601 tcaggctccc ttatacacag ccagtctgca ggtcgaccat agtgactgga tatgttgtgt
     3661 tttacagtat tatgtagtct gttttttatg caaaatctaa tttaatatat tgatatttat
     3721 atcattttac gtttctcgtt cagctttctt gtacaaagtg gttgatgatc ctaggtgagt
     3781 ctagagagtt aattaagacc cgggactagt ccctagagtc ctgctttaat gagatatgcg
     3841 agacgcctat gatcgcatga tatttgcttt caattctgtt gtgcacgttg taaaaaacct
     3901 gagcatgtgt agctcagatc cttaccgccg gtttcggttc attctaatga atatatcacc
     3961 cgttactatc gtatttttat gaataatatt ctccgttcaa tttactgatt gtaccctact
     4021 acttatatgt acaatattaa aatgaaaaca atatattgtg ctgaataggt ttatagcgac
     4081 atctatgata gagcgccaca ataacaaaca attgcgtttt attattacaa atccaatttt
     4141 aaaaaaagcg gcagaaccgg tcaaacctaa aagactgatt acataaatct tattcaaatt
     4201 tcaaaagtgc cccaggggct agtatctacg acacaccgag cggcgaacta ataacgctca
     4261 ctgaagggaa ctccggttcc ccgccggcgc gcatgggtga gattccttga agttgagtat
     4321 tggccgtccg ctctaccgaa agttacgggc accattcaac ccggtccagc acggcggccg
     4381 ggtaaccgac ttgctgcccc gagaattatg cagcattttt ttggtgtatg tgggccccaa
     4441 atgaagtgca ggtcaaacct tgacagtgac gacaaatcgt tgggcgggtc cagggcgaat
     4501 tttgcgacaa catgtcgagg ctcagcagga cctgcaggtc gactctagag gatccccggg
     4561 taccgagctc gaattcgtaa tcatgtcata gctgtttcct gtgtgaaatt gttatccgct
     4621 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     4681 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     4741 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     4801 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     4861 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     4921 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     4981 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     5041 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     5101 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     5161 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     5221 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     5281 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     5341 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     5401 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     5461 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     5521 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     5581 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     5641 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     5701 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     5761 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     5821 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     5881 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     5941 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     6001 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     6061 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     6121 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     6181 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     6241 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     6301 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     6361 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     6421 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     6481 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     6541 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     6601 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     6661 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     6721 ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa
     6781 taggcgtatc acgaggccct ttcgtc
//
