LOCUS       sequence-447312-        8359 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty Kanamycin resistant GreenGate destination vector based on 
            pGGP-AG. Domesticated of Esp3I sites in the backbone and 
            chloramphenicol resistance gene..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8359)
  AUTHORS   Pennetti VJ, LaFayette PR, Parrott WA
  TITLE     MultiGreen: A multiplexing architecture for GreenGate cloning.
  JOURNAL   PLoS One. 2024 Sep 18;19(9):e0306008. doi: 
            10.1371/journal.pone.0306008. eCollection 2024.
  PUBMED    39292669
REFERENCE   2  (bases 1 to 8359)
  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: "2024-09-18"; volume: "19(9):e0306008. doi"; pages: " 
            10.1371/journal.pone.0306008. eCollection 2024"
COMMENT     Sequence Label: pVP077
FEATURES             Location/Qualifiers
     source          1..8359
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     133..152
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     217..236
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     misc_feature    518..542
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     promoter        join(699..704,705..722,723..729)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             783..1442
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFLVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(849..868)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1784..2089
                     /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     1968..1987
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     misc_feature    2169..2193
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             3524..4153
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             4582..5655
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
                     QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
                     KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
                     GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
                     ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
                     LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
                     LVMRYRNLIEGEASAGS"
     rep_origin      5721..5915
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(6151..6173)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    6259..6399
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6314..6333
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6414..6431)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6585..7173)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6665..6684)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7769..8359,1..225))
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(join(7769..8359,1..225))
                     /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="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
ORIGIN
        1 cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg
       61 tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga
      121 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
      181 taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt
      241 actgtttatg taagcagaca gttttattgt tcatgatctg gatcacaggc agcaacgctc
      301 tgtcatcgtt acaatcaaca tgctaccctc cgcgagatca tccgtgtttc aaacccggca
      361 gcttagttgc cgttcttccg aatagcatcg gtaacatgag caaagtctgc cgccttacaa
      421 cggctctccc gctgacgccg tcccggactg atgggctgcc tgtatcgagt ggtgattttg
      481 tgccgagctg ccggtcgggg agctgttggc tggctggtgg caggatatat tgtggtgtaa
      541 acaaattgac gcttagacaa cttaataaca cattgcggac gtttttaatg tactgaatta
      601 acgccgaatt gaattcgcgg tacccgggga tcctctagag ggcccgacgt cgcatgcctg
      661 cagatactga gaccgcggcc gcattaggca ccccaggctt tacactttat gcttccggct
      721 cgtataatgt gtggattttg agttaggatc cgtcgagatt ttcaggagct aaggaagcta
      781 aaatggagaa aaaaatcact ggatatacca ccgttgatat atcccaatgg catcgtaaag
      841 aacattttga ggcatttcag tcagttgctc aatgtaccta taaccagacc gttcagctgg
      901 atattacggc ctttttaaag accgtaaaga aaaataagca caagttttat ccggccttta
      961 ttcacattct tgcccgcctg atgaatgctc atccggaatt ccgtatggca atgaaagacg
     1021 gtgagctggt gatatgggat agtgttcacc cttgttacac cgttttccat gagcaaactg
     1081 aaacgttttc atcgctctgg agtgaatacc acgacgattt ccggcagttt ctacacatat
     1141 attcgcaaga tgtggcgtgt tacggtgaaa acctggccta tttccctaaa gggtttattg
     1201 agaatatgtt tttagtctca gccaatccct gggtgagttt caccagtttt gatttaaacg
     1261 tggccaatat ggacaacttc ttcgcccccg ttttcaccat gggcaaatat tatacgcaag
     1321 gcgacaaggt gctgatgccg ctggcgattc aggttcatca tgccgtttgt gatggcttcc
     1381 atgtcggcag aatgcttaat gaattacaac agtactgcga tgagtggcag ggcggggcgt
     1441 aaacgcgtgg atccggctta ctaaaagcca gataacagta tgcgtatttg cgcgctgatt
     1501 tttgcggtat aagaatatat actgatatgt atacccgaag tatgtcaaaa agaggtatgc
     1561 tatgaagcag cgtattacag tgacagttga cagcgacagc tatcagttgc tcaaggcata
     1621 tatgatgtca atatctccgg tctggtaagc acaaccatgc agaatgaagc ccgtcgtctg
     1681 cgtgccgaac gctggaaagc ggaaaatcag gaagggatgg ctgaggtcgc ccggtttatt
     1741 gaaatgaacg gctcttttgc tgacgagaac aggggctggt gaaatgcagt ttaaggttta
     1801 cacctataaa agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga
     1861 cacgcccggg cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt
     1921 ctcccgtgaa ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac
     1981 cgatatggcc agtgtgccgg tttccgttat cggggaagaa gtggctgatc tcagccaccg
     2041 cgaaaatgac atcaaaaacg ccattaacct gatgttctgg ggaatataag tcgacggtct
     2101 caaggtaagc ttagcttgag cttggatcag attgtcgttt cccgccttca gtttaaacta
     2161 tcagtgtttg acaggatata ttggcgggta aacctaagag aaaagagcgt ttattagaat
     2221 aatcggatat ttaaaagggc gtgaaaaggt ttatccgttc gtccatttgt atgtgcatgc
     2281 caaccacagg gttcccctcg ggatcaaagt actttaaagt actttaaagt actttaaagt
     2341 actttgatcc aacccctccg ctgctatagt gcagtcggct tctgacgttc agtgcagccc
     2401 tcttctgaaa acgacatgtc gcacaagtcc taagttacgc gacaggctgc cgccctgccc
     2461 ttttcctggc gttttcttgt cgcgtgtttt agtcgcataa agtagaatac ttgcgactag
     2521 aaccggagac attacgccat gaacaagagc gccgccgctg gcctgctggg ctatgcccgc
     2581 gtcagcaccg acgaccagga cttgaccaac caacgggccg aactgcacgc ggccggctgc
     2641 accaagctgt tttccgagaa gatcaccggc accaggcgcg accgcccgga gctggccagg
     2701 atgcttgacc acctacgccc tggcgacgtt gtgacagtga ccaggctaga ccgcctggcc
     2761 cgcagcaccc gcgacctact ggacattgcc gagcgcatcc aggaggccgg cgcgggcctg
     2821 cgtagcctgg cagagccgtg ggccgacacc accacgccgg ccggccgcat ggtgttgacc
     2881 gtgttcgccg gcattgccga gttcgagcgt tccctaatca tcgaccgcac ccggagcggg
     2941 cgcgaggccg ccaaggcccg aggcgtgaag tttggccccc gccctaccct caccccggca
     3001 cagatcgcgc acgcccgcga gctgatcgac caggaaggcc gcaccgtgaa agaggcggct
     3061 gcactgcttg gcgtgcatcg ctcgaccctg taccgcgcac ttgagcgcag cgaggaagtg
     3121 acgcccaccg aggccaggcg gcgcggtgcc ttccgtgagg acgcattgac cgaggccgac
     3181 gccctggcgg ccgccgagaa tgaacgccaa gaggaacaag catgaaaccg caccaggacg
     3241 gccaggacga accgtttttc attaccgaag agatcgaggc ggagatgatc ccggccgggt
     3301 acgtgttcga gccgcccgcg cacgtatcaa ccgtgcggct gcatgaaatc ctggccggtt
     3361 tgtctgatgc caagctggcg gcctggccgg ccagcttggc cgctgaagaa accgagcgcc
     3421 gccgtctaaa aaggtgatgt gtatttgagt aaaacagctt gcgtcatgcg gtcgctgcgt
     3481 atatgatgcg atgagtaaat aaacaaatac gcaaggggaa cgcatgaagg ttatcgctgt
     3541 acttaaccag aaaggcgggt caggcaagac gaccatcgca acccatctag cccgcgccct
     3601 gcaactcgcc ggggccgatg ttctgttagt cgattccgat ccccagggca gtgcccgcga
     3661 ttgggcggcc gtgcgggaag atcaaccgct aaccgttgtc ggcatcgacc gcccgacgat
     3721 tgaccgcgac gtgaaggcca tcggccggcg cgacttcgta gtgatcgacg gagcgcccca
     3781 ggcggcggac ttggctgtgt ccgcgatcaa ggcagccgac ttcgtgctga ttccggtgca
     3841 gccaagccct tacgacatat gggccaccgc cgacctggtg gagctggtta agcagcgcat
     3901 tgaggtcacg gatggaaggc tacaagcggc ctttgtcgtg tcgcgggcga tcaaaggcac
     3961 gcgcatcggc ggtgaggttg ccgaggcgct ggccgggtac gagctgccca ttcttgagtc
     4021 ccgtatcacg cagcgcgtga gctacccagg cactgccgcc gccggcacaa ccgttcttga
     4081 atcagaaccc gagggcgacg ctgcccgcga ggtccaggcg ctggccgctg aaattaaatc
     4141 aaaactcatt tgagttaatg aggtaaagag aaaatgagca aaagcacaaa cacgctaagt
     4201 gccggccgtc cgagcgcacg cagcagcaag gctgcaacgt tggccagcct ggcagacacg
     4261 ccagccatga agcgggtcaa ctttcagttg ccggcggagg atcacaccaa gctgaagatg
     4321 tacgcggtac gccaaggcaa gaccattacc gagctgctat ctgaatacat cgcgcagcta
     4381 ccagagtaaa tgagcaaatg aataaatgag tagatgaatt ttagcggcta aaggaggcgg
     4441 catggaaaat caagaacaac caggcaccga cgccgtggaa tgccccatgt gtggaggaac
     4501 gggcggttgg ccaggcgtaa gcggctgggt tgtctgccgg ccctgcaatg gcactggaac
     4561 ccccaagccc gaggaatcgg cgtgagcggt cgcaaaccat ccggcccggt acaaatcggc
     4621 gcggcgctgg gtgatgacct ggtggagaag ttgaaggccg cgcaggccgc ccagcggcaa
     4681 cgcatcgagg cagaagcacg ccccggtgaa tcgtggcaag cggccgctga tcgaatccgc
     4741 aaagaatccc ggcaaccgcc ggcagccggt gcgccgtcga ttaggaagcc gcccaagggc
     4801 gacgagcaac cagatttttt cgttccgatg ctctatgacg tgggcacccg cgatagtcgc
     4861 agcatcatgg acgtggccgt tttccgtctg tcgaagcgtg accgacgagc tggcgaggtg
     4921 atccgctacg agcttccaga cgggcacgta gaggtttccg cagggccggc cggcatggcc
     4981 agtgtgtggg attacgacct ggtactgatg gcggtttccc atctaaccga atccatgaac
     5041 cgataccggg aagggaaggg agacaagccc ggccgcgtgt tccgtccaca cgttgcggac
     5101 gtactcaagt tctgccggcg agccgatggc ggaaagcaga aagacgacct ggtagaaacc
     5161 tgcattcggt taaacaccac gcacgttgcc atgcagcgta cgaagaaggc caagaacggc
     5221 cgcctggtga cggtatccga gggtgaagcc ttgattagcc gctacaagat cgtaaagagc
     5281 gaaaccgggc ggccggagta catcgagatc gagctagctg attggatgta ccgcgagatc
     5341 acagaaggca agaacccgga cgtgctgacg gttcaccccg attacttttt gatcgatccc
     5401 ggcatcggcc gttttctcta ccgcctggca cgccgcgccg caggcaaggc agaagccaga
     5461 tggttgttca agacgatcta cgaacgcagt ggcagcgccg gagagttcaa gaagttctgt
     5521 ttcaccgtgc gcaagctgat cgggtcaaat gacctgccgg agtacgattt gaaggaggag
     5581 gcggggcagg ctggcccgat cctagtcatg cgctaccgca acctgatcga gggcgaagca
     5641 tccgccggtt cctaatgtac ggagcagatg ctagggcaaa ttgccctagc aggggaaaaa
     5701 ggtcgaaaag ctctctttcc tgtggatagc acgtacattg ggaacccaaa gccgtacatt
     5761 gggaaccgga acccgtacat tgggaaccca aagccgtaca ttgggaaccg gtcacacatg
     5821 taagtgactg atataaaaga gaaaaaaggc gatttttccg cctaaaactc tttaaaactt
     5881 attaaaactc ttaaaacccg cctggcctgt gcataactgt ctggccagcg cacagccgaa
     5941 gagctgcaaa aagcgcctac ccttcggtcg ctgcgctccc tacgccccgc cgcttcgcgt
     6001 cggcctatcg cggccgctgg ccgctcaaaa atggctggcc tacggccagg caatctacca
     6061 gggcgcggac aagccgcgcc gtcgccactc gaccgccggc gcccacatca aggcaccctg
     6121 cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagccggt
     6181 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     6241 tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac
     6301 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa
     6361 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc
     6421 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     6481 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     6541 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     6601 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     6661 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     6721 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     6781 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     6841 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     6901 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     6961 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     7021 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     7081 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     7141 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     7201 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgca tgatatatct
     7261 cccaatttgt gtagggctta ttatgcacgc ttaaaaataa taaaagcaga cttgacctga
     7321 tagtttggct gtgagcaatt atgtgcttag tgcatctaac gcttgagtta agccgcgccg
     7381 cgaagcggcg tcggcttgaa cgaatttcta gctagacatt atttgccgac taccttggtg
     7441 atctcgcctt tcacgtagtg gacaaattct tccaactgat ctgcgcgcga ggccaagcga
     7501 tcttcttctt gtccaagata agcctgtcta gcttcaagta tgacgggctg atactgggcc
     7561 ggcaggcgct ccattgccca gtcggcagcg acatccttcg gcgcgatttt gccggttact
     7621 gcgctgtacc aaatgcggga caacgtaagc actacatttc gctcatcgcc agcccagtcg
     7681 ggcggcgagt tccatagcgt taaggtttca tttagcgcct caaatagatc ctgttcagga
     7741 accggatcaa agagttcctc cgccgctgtt agaaaaactc atcgagcatc aaatgaaact
     7801 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     7861 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     7921 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     7981 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca
     8041 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     8101 caaccaaacc gttattcatt cgtgattgcg cctgagcgag ccgaaatacg cgatcgctgt
     8161 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     8221 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
     8281 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     8341 gaagaggcat aaattccgt
//
