LOCUS       sequence-420613-        7523 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Gateway cloning compatible vector for transient expression in 
            protoplasts. Arabidopsis UBQ10 promoter for N-terminal mVenus fusion
            protein. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7523)
  AUTHORS   Allen J, Flanagan M, Pathak S, Emenecker S, Emenecker R, Strader L
  TITLE     An Improved Toolkit of Gateway- and Gibson Assembly-Compatible 
            Vectors for Protoplast Transfection and Agrobacterium-Mediated Plant
            Transformation.
  JOURNAL   Res Sq. 2023 Nov 7:rs.3.rs-3558280. doi: 
            10.21203/rs.3.rs-3558280/v1. Preprint.
  PUBMED    37986897
REFERENCE   2  (bases 1 to 7523)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Res Sq. 
            2023 Nov 7:rs.3.rs-3558280. doi: 10.21203/rs.3.rs-3558280/v1. 
            Preprint."
COMMENT     Sequence Label: pLCS101
FEATURES             Location/Qualifiers
     source          1..7523
                     /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"
     CDS             join(1989..1991,1992..1994,1995..2705)
                     /codon_start=1
                     /product="enhanced YFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2034..2055)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2295..2314)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2642..2663
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    2775..2899
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(2924..2929,2930..2947,2948..2954)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             3008..3667
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3074..3093)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             4009..4314
                     /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     4193..4212
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(4355..4479)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     terminator      4538..5245
                     /label=OCS terminator
                     /note="octopine synthase terminator"
     primer_bind     complement(5302..5318)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5302..5318)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5315..5337)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5326..5342
                     /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(5350..5356,5357..5374,5375..5380))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5395..5416
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5533..5550)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5704..6292)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5784..5803)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6463..7254,7255..7323))
                     /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     7086..7105
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7324..7428)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7496..7514
                     /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 tcctcgagat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     2041 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     2101 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     2161 ggcccaccct cgtgaccacc ttcggctacg gcctgcagtg cttcgcccgc taccccgacc
     2221 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     2281 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     2341 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     2401 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
     2461 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     2521 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     2581 acaaccacta cctgagctac cagtccaaac tgagcaaaga ccccaacgag aagcgcgatc
     2641 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     2701 acaagtccgg actcagatca cgagctcaag cttcaaattc tgcagtcgac ggtaccgcgg
     2761 gcccgggatc atcaacaagt ttgtacaaaa aagctgaacg agaaacgtaa aatgatataa
     2821 atatcaatat attaaattag attttgcata aaaaacagac tacataatac tgtaaaacac
     2881 aacatatcca gtcactatgg cggccgcatt aggcacccca ggctttacac tttatgcttc
     2941 cggctcgtat aatgtgtgga ttttgagtta ggatccgtcg agattttcag gagctaagga
     3001 agctaaaatg gagaaaaaaa tcactggata taccaccgtt gatatatccc aatggcatcg
     3061 taaagaacat tttgaggcat ttcagtcagt tgctcaatgt acctataacc agaccgttca
     3121 gctggatatt acggcctttt taaagaccgt aaagaaaaat aagcacaagt tttatccggc
     3181 ctttattcac attcttgccc gcctgatgaa tgctcatccg gagttccgta tggcaatgaa
     3241 agacggtgag ctggtgatat gggatagtgt tcacccttgt tacaccgttt tccatgagca
     3301 aactgaaacg ttttcatcgc tctggagtga ataccacgac gatttccggc agtttctaca
     3361 catatattcg caagatgtgg cgtgttacgg tgaaaacctg gcctatttcc ctaaagggtt
     3421 tattgagaat atgtttttcg tctcagccaa tccctgggtg agtttcacca gttttgattt
     3481 aaacgtggcc aatatggaca acttcttcgc ccccgttttc accatgggca aatattatac
     3541 gcaaggcgac aaggtgctga tgccgctggc gattcaggtt catcatgccg tttgtgatgg
     3601 cttccatgtc ggcagaatgc ttaatgaatt acaacagtac tgcgatgagt ggcagggcgg
     3661 ggcgtaaaga tctggatccg gcttactaaa agccagataa cagtatgcgt atttgcgcgc
     3721 tgatttttgc ggtataagaa tatatactga tatgtatacc cgaagtatgt caaaaagagg
     3781 tatgctatga agcagcgtat tacagtgaca gttgacagcg acagctatca gttgctcaag
     3841 gcatatatga tgtcaatatc tccggtctgg taagcacaac catgcagaat gaagcccgtc
     3901 gtctgcgtgc cgaacgctgg aaagcggaaa atcaggaagg gatggctgag gtcgcccggt
     3961 ttattgaaat gaacggctct tttgctgacg agaacagggg ctggtgaaat gcagtttaag
     4021 gtttacacct ataaaagaga gagccgttat cgtctgtttg tggatgtaca gagtgatatt
     4081 attgacacgc ccgggcgacg gatggtgatc cccctggcca gtgcacgtct gctgtcagat
     4141 aaagtctccc gtgaacttta cccggtggtg catatcgggg atgaaagctg gcgcatgatg
     4201 accaccgata tggccagtgt gccggtctcc gttatcgggg aagaagtggc tgatctcagc
     4261 caccgcgaaa atgacatcaa aaacgccatt aacctgatgt tctggggaat ataaatgtca
     4321 ggctccctta tacacagcca gtctgcaggt cgaccatagt gactggatat gttgtgtttt
     4381 acagtattat gtagtctgtt ttttatgcaa aatctaattt aatatattga tatttatatc
     4441 attttacgtt tctcgttcag ctttcttgta caaagtggtt gatgatccta ggtgagtcta
     4501 gagagttaat taagacccgg gactagtccc tagagtcctg ctttaatgag atatgcgaga
     4561 cgcctatgat cgcatgatat ttgctttcaa ttctgttgtg cacgttgtaa aaaacctgag
     4621 catgtgtagc tcagatcctt accgccggtt tcggttcatt ctaatgaata tatcacccgt
     4681 tactatcgta tttttatgaa taatattctc cgttcaattt actgattgta ccctactact
     4741 tatatgtaca atattaaaat gaaaacaata tattgtgctg aataggttta tagcgacatc
     4801 tatgatagag cgccacaata acaaacaatt gcgttttatt attacaaatc caattttaaa
     4861 aaaagcggca gaaccggtca aacctaaaag actgattaca taaatcttat tcaaatttca
     4921 aaagtgcccc aggggctagt atctacgaca caccgagcgg cgaactaata acgctcactg
     4981 aagggaactc cggttccccg ccggcgcgca tgggtgagat tccttgaagt tgagtattgg
     5041 ccgtccgctc taccgaaagt tacgggcacc attcaacccg gtccagcacg gcggccgggt
     5101 aaccgacttg ctgccccgag aattatgcag catttttttg gtgtatgtgg gccccaaatg
     5161 aagtgcaggt caaaccttga cagtgacgac aaatcgttgg gcgggtccag ggcgaatttt
     5221 gcgacaacat gtcgaggctc agcaggacct gcaggtcgac tctagaggat ccccgggtac
     5281 cgagctcgaa ttcgtaatca tgtcatagct gtttcctgtg tgaaattgtt atccgctcac
     5341 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     5401 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     5461 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     5521 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     5581 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     5641 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     5701 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     5761 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     5821 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     5881 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     5941 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     6001 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     6061 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     6121 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     6181 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     6241 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     6301 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     6361 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     6421 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     6481 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     6541 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     6601 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     6661 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     6721 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     6781 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     6841 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     6901 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     6961 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     7021 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     7081 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     7141 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     7201 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     7261 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     7321 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     7381 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     7441 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
     7501 gcgtatcacg aggccctttc gtc
//
