LOCUS       sequence-420620-        7697 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway cloning compatible vector for transient expression in 
            protoplasts. Arabidopsis UBQ10 promoter for N-terminal HaloTag 
            fusion protein..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7697)
  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 7697)
  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: pLCS104
FEATURES             Location/Qualifiers
     source          1..7697
                     /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             1989..2879
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     protein_bind    2949..3073
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(3098..3103,3104..3121,3122..3128)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             3182..3841
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3248..3267)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             4183..4488
                     /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     4367..4386
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(4529..4653)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     terminator      4712..5419
                     /label=OCS terminator
                     /note="octopine synthase terminator"
     primer_bind     complement(5476..5492)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5476..5492)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5489..5511)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5500..5516
                     /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(5524..5530,5531..5548,5549..5554))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5569..5590
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5707..5724)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5878..6466)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5958..5977)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6637..7428,7429..7497))
                     /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     7260..7279
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7498..7602)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7670..7688
                     /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 ggcagaaatc ggtactggct ttccattcga cccccattat gtggaagtcc
     2041 tgggcgagcg catgcactac gtcgatgttg gtccgcgcga tggcacccct gtgctgttcc
     2101 tgcacggtaa cccgacctcc tcctacgtgt ggcgcaacat catcccgcat gttgcaccga
     2161 cccatcgctg cattgctcca gacctgatcg gtatgggcaa atccgacaaa ccagacctgg
     2221 gttatttctt cgacgaccac gtccgcttca tggatgcctt catcgaagcc ctgggtctgg
     2281 aagaggtcgt cctggtcatt cacgactggg gctccgctct gggtttccac tgggccaagc
     2341 gcaatccaga gcgcgtcaaa ggtattgcat ttatggagtt catccgccct atcccgacct
     2401 gggacgaatg gccagaattt gcccgcgaga ccttccaggc cttccgcacc accgacgtcg
     2461 gccgcaagct gatcatcgat cagaacgttt ttatcgaggg tacgctgccg atgggtgtcg
     2521 tccgcccgct gactgaagtc gagatggacc attaccgcga gccgttcctg aatcctgttg
     2581 accgcgagcc actgtggcgc ttcccaaacg agctgccaat cgccggtgag ccagcgaaca
     2641 tcgtcgcgct ggtcgaagaa tacatggact ggctgcacca gtcccctgtc ccgaagctgc
     2701 tgttctgggg caccccaggc gttctgatcc caccggccga agccgctcgc ctggccaaaa
     2761 gcctgcctaa ctgcaaggct gtggacatcg gcccgggtct gaatctgctg caagaagaca
     2821 acccggacct gatcggcagc gagatcgcgc gctggctgtc gacgcttgag atttccggct
     2881 ccggactcag atcacgagct caagcttcaa attctgcagt cgacggtacc gcgggcccgg
     2941 gatcatcaac aagtttgtac aaaaaagctg aacgagaaac gtaaaatgat ataaatatca
     3001 atatattaaa ttagattttg cataaaaaac agactacata atactgtaaa acacaacata
     3061 tccagtcact atggcggccg cattaggcac cccaggcttt acactttatg cttccggctc
     3121 gtataatgtg tggattttga gttaggatcc gtcgagattt tcaggagcta aggaagctaa
     3181 aatggagaaa aaaatcactg gatataccac cgttgatata tcccaatggc atcgtaaaga
     3241 acattttgag gcatttcagt cagttgctca atgtacctat aaccagaccg ttcagctgga
     3301 tattacggcc tttttaaaga ccgtaaagaa aaataagcac aagttttatc cggcctttat
     3361 tcacattctt gcccgcctga tgaatgctca tccggagttc cgtatggcaa tgaaagacgg
     3421 tgagctggtg atatgggata gtgttcaccc ttgttacacc gttttccatg agcaaactga
     3481 aacgttttca tcgctctgga gtgaatacca cgacgatttc cggcagtttc tacacatata
     3541 ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat ttccctaaag ggtttattga
     3601 gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc accagttttg atttaaacgt
     3661 ggccaatatg gacaacttct tcgcccccgt tttcaccatg ggcaaatatt atacgcaagg
     3721 cgacaaggtg ctgatgccgc tggcgattca ggttcatcat gccgtttgtg atggcttcca
     3781 tgtcggcaga atgcttaatg aattacaaca gtactgcgat gagtggcagg gcggggcgta
     3841 aagatctgga tccggcttac taaaagccag ataacagtat gcgtatttgc gcgctgattt
     3901 ttgcggtata agaatatata ctgatatgta tacccgaagt atgtcaaaaa gaggtatgct
     3961 atgaagcagc gtattacagt gacagttgac agcgacagct atcagttgct caaggcatat
     4021 atgatgtcaa tatctccggt ctggtaagca caaccatgca gaatgaagcc cgtcgtctgc
     4081 gtgccgaacg ctggaaagcg gaaaatcagg aagggatggc tgaggtcgcc cggtttattg
     4141 aaatgaacgg ctcttttgct gacgagaaca ggggctggtg aaatgcagtt taaggtttac
     4201 acctataaaa gagagagccg ttatcgtctg tttgtggatg tacagagtga tattattgac
     4261 acgcccgggc gacggatggt gatccccctg gccagtgcac gtctgctgtc agataaagtc
     4321 tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc
     4381 gatatggcca gtgtgccggt ctccgttatc ggggaagaag tggctgatct cagccaccgc
     4441 gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataaat gtcaggctcc
     4501 cttatacaca gccagtctgc aggtcgacca tagtgactgg atatgttgtg ttttacagta
     4561 ttatgtagtc tgttttttat gcaaaatcta atttaatata ttgatattta tatcatttta
     4621 cgtttctcgt tcagctttct tgtacaaagt ggttgatgat cctaggtgag tctagagagt
     4681 taattaagac ccgggactag tccctagagt cctgctttaa tgagatatgc gagacgccta
     4741 tgatcgcatg atatttgctt tcaattctgt tgtgcacgtt gtaaaaaacc tgagcatgtg
     4801 tagctcagat ccttaccgcc ggtttcggtt cattctaatg aatatatcac ccgttactat
     4861 cgtattttta tgaataatat tctccgttca atttactgat tgtaccctac tacttatatg
     4921 tacaatatta aaatgaaaac aatatattgt gctgaatagg tttatagcga catctatgat
     4981 agagcgccac aataacaaac aattgcgttt tattattaca aatccaattt taaaaaaagc
     5041 ggcagaaccg gtcaaaccta aaagactgat tacataaatc ttattcaaat ttcaaaagtg
     5101 ccccaggggc tagtatctac gacacaccga gcggcgaact aataacgctc actgaaggga
     5161 actccggttc cccgccggcg cgcatgggtg agattccttg aagttgagta ttggccgtcc
     5221 gctctaccga aagttacggg caccattcaa cccggtccag cacggcggcc gggtaaccga
     5281 cttgctgccc cgagaattat gcagcatttt tttggtgtat gtgggcccca aatgaagtgc
     5341 aggtcaaacc ttgacagtga cgacaaatcg ttgggcgggt ccagggcgaa ttttgcgaca
     5401 acatgtcgag gctcagcagg acctgcaggt cgactctaga ggatccccgg gtaccgagct
     5461 cgaattcgta atcatgtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     5521 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     5581 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     5641 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     5701 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     5761 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     5821 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     5881 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     5941 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     6001 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     6061 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     6121 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     6181 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     6241 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     6301 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     6361 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     6421 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     6481 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     6541 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     6601 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     6661 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     6721 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     6781 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     6841 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     6901 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     6961 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     7021 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     7081 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     7141 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     7201 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     7261 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     7321 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     7381 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     7441 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     7501 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     7561 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     7621 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat
     7681 cacgaggccc tttcgtc
//
