LOCUS       sequence-440976-        8381 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MultiGreen 2.0 BC level 1 acceptor vector, kanamycin selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8381)
  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 8381)
  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: pVP312
FEATURES             Location/Qualifiers
     source          1..8381
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     448..467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     701..718
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    733..873
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(799..818)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     959..981
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     rep_origin      1217..1411
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     CDS             complement(1477..2550)
                     /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"
     CDS             complement(2979..3608)
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     misc_feature    4939..4963
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(5054..5359)
                     /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     complement(5156..5175)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(5701..6360)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFLVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6275..6294
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(6414..6420,6421..6438,6439..6444))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    6612..6636
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     primer_bind     complement(6918..6937)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6929..7744
                     /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             6929..7744
                     /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"
     primer_bind     complement(7002..7021)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(8340..8381,1..547)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
       61 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
      121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      241 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      301 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      361 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      421 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      481 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      541 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      601 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
      661 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
      721 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
      781 catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
      841 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
      901 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccggctcc
      961 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagggtgcc
     1021 ttgatgtggg cgccggcggt cgagtggcga cggcgcggct tgtccgcgcc ctggtagatt
     1081 gcctggccgt aggccagcca tttttgagcg gccagcggcc gcgataggcc gacgcgaagc
     1141 ggcggggcgt agggagcgca gcgaccgaag ggtaggcgct ttttgcagct cttcggctgt
     1201 gcgctggcca gacagttatg cacaggccag gcgggtttta agagttttaa taagttttaa
     1261 agagttttag gcggaaaaat cgcctttttt ctcttttata tcagtcactt acatgtgtga
     1321 ccggttccca atgtacggct ttgggttccc aatgtacggg ttccggttcc caatgtacgg
     1381 ctttgggttc ccaatgtacg tgctatccac aggaaagaga gcttttcgac ctttttcccc
     1441 tgctagggca atttgcccta gcatctgctc cgtacattag gaaccggcgg atgcttcgcc
     1501 ctcgatcagg ttgcggtagc gcatgactag gatcgggcca gcctgccccg cctcctcctt
     1561 caaatcgtac tccggcaggt catttgaccc gatcagcttg cgcacggtga aacagaactt
     1621 cttgaactct ccggcgctgc cactgcgttc gtagatcgtc ttgaacaacc atctggcttc
     1681 tgccttgcct gcggcgcggc gtgccaggcg gtagagaaaa cggccgatgc cgggatcgat
     1741 caaaaagtaa tcggggtgaa ccgtcagcac gtccgggttc ttgccttctg tgatctcgcg
     1801 gtacatccaa tcagctagct cgatctcgat gtactccggc cgcccggttt cgctctttac
     1861 gatcttgtag cggctaatca aggcttcacc ctcggatacc gtcaccaggc ggccgttctt
     1921 ggccttcttc gtacgctgca tggcaacgtg cgtggtgttt aaccgaatgc aggtttctac
     1981 caggtcgtct ttctgctttc cgccatcggc tcgccggcag aacttgagta cgtccgcaac
     2041 gtgtggacgg aacacgcggc cgggcttgtc tcccttccct tcccggtatc ggttcatgga
     2101 ttcggttaga tgggaaaccg ccatcagtac caggtcgtaa tcccacacac tggccatgcc
     2161 ggccggccct gcggaaacct ctacgtgccc gtctggaagc tcgtagcgga tcacctcgcc
     2221 agctcgtcgg tcacgcttcg acagacggaa aacggccacg tccatgatgc tgcgactatc
     2281 gcgggtgccc acgtcataga gcatcggaac gaaaaaatct ggttgctcgt cgcccttggg
     2341 cggcttccta atcgacggcg caccggctgc cggcggttgc cgggattctt tgcggattcg
     2401 atcagcggcc gcttgccacg attcaccggg gcgtgcttct gcctcgatgc gttgccgctg
     2461 ggcggcctgc gcggccttca acttctccac caggtcatca cccagcgccg cgccgatttg
     2521 taccgggccg gatggtttgc gaccgctcac gccgattcct cgggcttggg ggttccagtg
     2581 ccattgcagg gccggcagac aacccagccg cttacgcctg gccaaccgcc cgttcctcca
     2641 cacatggggc attccacggc gtcggtgcct ggttgttctt gattttccat gccgcctcct
     2701 ttagccgcta aaattcatct actcatttat tcatttgctc atttactctg gtagctgcgc
     2761 gatgtattca gatagcagct cggtaatggt cttgccttgg cgtaccgcgt acatcttcag
     2821 cttggtgtga tcctccgccg gcaactgaaa gttgacccgc ttcatggctg gcgtgtctgc
     2881 caggctggcc aacgttgcag ccttgctgct gcgtgcgctc ggacggccgg cacttagcgt
     2941 gtttgtgctt ttgctcattt tctctttacc tcattaactc aaatgagttt tgatttaatt
     3001 tcagcggcca gcgcctggac ctcgcgggca gcgtcgccct cgggttctga ttcaagaacg
     3061 gttgtgccgg cggcggcagt gcctgggtag ctcacgcgct gcgtgatacg ggactcaaga
     3121 atgggcagct cgtacccggc cagcgcctcg gcaacctcac cgccgatgcg cgtgcctttg
     3181 atcgcccgcg acacgacaaa ggccgcttgt agccttccat ccgtgacctc aatgcgctgc
     3241 ttaaccagct ccaccaggtc ggcggtggcc catatgtcgt aagggcttgg ctgcaccgga
     3301 atcagcacga agtcggctgc cttgatcgcg gacacagcca agtccgccgc ctggggcgct
     3361 ccgtcgatca ctacgaagtc gcgccggccg atggccttca cgtcgcggtc aatcgtcggg
     3421 cggtcgatgc cgacaacggt tagcggttga tcttcccgca cggccgccca atcgcgggca
     3481 ctgccctggg gatcggaatc gactaacaga acatcggccc cggcgagttg cagggcgcgg
     3541 gctagatggg ttgcgatggt cgtcttgcct gacccgcctt tctggttaag tacagcgata
     3601 accttcatgc gttccccttg cgtatttgtt tatttactca tcgcatcata tacgcagcga
     3661 ccgcatgacg caagctgttt tactcaaata cacatcacct ttttagacgg cggcgctcgg
     3721 tttcttcagc ggccaagctg gccggccagg ccgccagctt ggcatcagac aaaccggcca
     3781 ggatttcatg cagccgcacg gttgatacgt gcgcgggcgg ctcgaacacg tacccggccg
     3841 ggatcatctc cgcctcgatc tcttcggtaa tgaaaaacgg ttcgtcctgg ccgtcctggt
     3901 gcggtttcat gcttgttcct cttggcgttc attctcggcg gccgccaggg cgtcggcctc
     3961 ggtcaatgcg tcctcacgga aggcaccgcg ccgcctggcc tcggtgggcg tcacttcctc
     4021 gctgcgctca agtgcgcggt acagggtcga gcgatgcacg ccaagcagtg cagccgcctc
     4081 tttcacggtg cggccttcct ggtcgatcag ctcgcgggcg tgcgcgatct gtgccggggt
     4141 gagggtaggg cgggggccaa acttcacgcc tcgggccttg gcggcctcgc gcccgctccg
     4201 ggtgcggtcg atgattaggg aacgctcgaa ctcggcaatg ccggcgaaca cggtcaacac
     4261 catgcggccg gccggcgtgg tggtgtcggc ccacggctct gccaggctac gcaggcccgc
     4321 gccggcctcc tggatgcgct cggcaatgtc cagtaggtcg cgggtgctgc gggccaggcg
     4381 gtctagcctg gtcactgtca caacgtcgcc agggcgtagg tggtcaagca tcctggccag
     4441 ctccgggcgg tcgcgcctgg tgccggtgat cttctcggaa aacagcttgg tgcagccggc
     4501 cgcgtgcagt tcggcccgtt ggttggtcaa gtcctggtcg tcggtgctga cgcgggcata
     4561 gcccagcagg ccagcggcgg cgctcttgtt catggcgtaa tgtctccggt tctagtcgca
     4621 agtattctac tttatgcgac taaaacacgc gacaagaaaa cgccaggaaa agggcagggc
     4681 ggcagcctgt cgcgtaactt aggacttgtg cgacatgtcg ttttcagaag agggctgcac
     4741 tgaacgtcag aagccgactg cactatagca gcggaggggt tggatcaaag tactttaaag
     4801 tactttaaag tactttaaag tactttgatc ccgaggggaa ccctgtggtt ggcatgcaca
     4861 tacaaatgga cgaacggata aaccttttca cgccctttta aatatccgat tattctaata
     4921 aacgctcttt tctcttaggt ttacccgcca atatatcctg tcaaacactg atagtttaaa
     4981 ctgaaggcgg gaaacgacaa tctgatccaa gctcaagcta agcttcgtct caaacaacct
     5041 tgagaccgtc gacttatatt ccccagaaca tcaggttaat ggcgtttttg atgtcatttt
     5101 cgcggtggct gagatcagcc acttcttccc cgataacgga aaccggcaca ctggccatat
     5161 cggtggtcat catgcgccag ctttcatccc cgatatgcac caccgggtaa agttcacggg
     5221 agactttatc tgacagcaga cgtgcactgg ccagggggat caccatccgt cgcccgggcg
     5281 tgtcaataat atcactctgt acatccacaa acagacgata acggctctct cttttatagg
     5341 tgtaaacctt aaactgcatt tcaccagccc ctgttctcgt cagcaaaaga gccgttcatt
     5401 tcaataaacc gggcgacctc agccatccct tcctgatttt ccgctttcca gcgttcggca
     5461 cgcagacgac gggcttcatt ctgcatggtt gtgcttacca gaccggagat attgacatca
     5521 tatatgcctt gagcaactga tagctgtcgc tgtcaactgt cactgtaata cgctgcttca
     5581 tagcatacct ctttttgaca tacttcgggt atacatatca gtatatattc ttataccgca
     5641 aaaatcagcg cgcaaatacg catactgtta tctggctttt agtaagccgg atccacgcgt
     5701 ttacgccccg ccctgccact catcgcagta ctgttgtaat tcattaagca ttctgccgac
     5761 atggaagcca tcacaaacgg catgatgaac ctgaatcgcc agcggcatca gcaccttgtc
     5821 gccttgcgta taatatttgc ccatggtgaa aacgggggcg aagaagttgt ccatattggc
     5881 cacgtttaaa tcaaaactgg tgaaactcac ccagggattg gctgagacta aaaacatatt
     5941 ctcaataaac cctttaggga aataggccag gttttcaccg taacacgcca catcttgcga
     6001 atatatgtgt agaaactgcc ggaaatcgtc gtggtattca ctccagagcg atgaaaacgt
     6061 ttcagtttgc tcatggaaaa cggtgtaaca agggtgaaca ctatcccata tcaccagctc
     6121 accgtctttc attgccatac ggaattccgg atgagcattc atcaggcggg caagaatgtg
     6181 aataaaggcc ggataaaact tgtgcttatt tttctttacg gtctttaaaa aggccgtaat
     6241 atccagctga acggtctggt tataggtaca ttgagcaact gactgaaatg cctcaaaatg
     6301 ttctttacga tgccattggg atatatcaac ggtggtatat ccagtgattt ttttctccat
     6361 tttagcttcc ttagctcctg aaaatctcga cggatcctaa ctcaaaatcc acacattata
     6421 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gcggccgcgg tctcagtatg
     6481 gctagagacg ctgcaggcat gcgacgtcgg gccctctaga ggatccccgg gtaccgcgaa
     6541 ttcaattcgg cgttaattca gtacattaaa aacgtccgca atgtgttatt aagttgtcta
     6601 agcgtcaatt tgtttacacc acaatatatc ctgccaccag ccagccaaca gctccccgac
     6661 cggcagctcg gcacaaaatc accactcgat acaggcagcc catcagtccg ggacggcgtc
     6721 agcgggagag ccgttgtaag gcggcagact ttgctcatgt taccgatgct attcggaaga
     6781 acggcaacta agctgccggg tttgaaacac ggatgatctc gcggagggta gcatgttgat
     6841 tgtaacgatg acagagcgtt gctgcctgtg atccagatca tgaacaataa aactgtctgc
     6901 ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcttgctc
     6961 taggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga
     7021 taatgtcggg caatcaggtg cgacaatcta tcgattgtat gggaagcccg atgcgccaga
     7081 gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag
     7141 actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc
     7201 tgatgatgca tggttactca ccactgcgat ccccgggaaa acagcattcc aggtattaga
     7261 agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt
     7321 gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc ggctcgctca
     7381 ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa
     7441 tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga
     7501 ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt
     7561 aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat
     7621 cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata
     7681 tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt
     7741 ctaacagcgg cggaggaact ctttgatccg gttcctgaac aggatctatt tgaggcgcta
     7801 aatgaaacct taacgctatg gaactcgccg cccgactggg ctggcgatga gcgaaatgta
     7861 gtgcttacgt tgtcccgcat ttggtacagc gcagtaaccg gcaaaatcgc gccgaaggat
     7921 gtcgctgccg actgggcaat ggagcgcctg ccggcccagt atcagcccgt catacttgaa
     7981 gctagacagg cttatcttgg acaagaagaa gatcgcttgg cctcgcgcgc agatcagttg
     8041 gaagaatttg tccactacgt gaaaggcgag atcaccaagg tagtcggcaa ataatgtcta
     8101 gctagaaatt cgttcaagcc gacgccgctt cgcggcgcgg cttaactcaa gcgttagatg
     8161 cactaagcac ataattgctc acagccaaac tatcaggtca agtctgcttt tattattttt
     8221 aagcgtgcat aataagccct acacaaattg ggagatatat catgcatgac caaaatccct
     8281 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     8341 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa c
//
