LOCUS       sequence-440970-        8239 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MultiGreen 2.0 DE level 1 acceptor vector, spectinomycin selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8239)
  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 8239)
  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: pVP081
FEATURES             Location/Qualifiers
     source          1..8239
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      434..1021
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     922..941
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1175..1192
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1207..1347
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1273..1292)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1433..1455
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     rep_origin      1691..1885
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     CDS             complement(1951..3024)
                     /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(3453..4082)
                     /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    5413..5437
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(5530..5835)
                     /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(5632..5651)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(6177..6836)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFLVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6751..6770
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(6890..6896,6897..6914,6915..6920))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    7091..7115
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     CDS             join(7636..8239,1..188)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
ORIGIN
        1 tcgcgccgaa ggatgtcgct gccgactggg caatggagcg cctgccggcc cagtatcagc
       61 ccgtcatact tgaagctaga caggcttatc ttggacaaga agaagatcgc ttggcctcgc
      121 gcgcagatca gttggaagaa tttgtccact acgtgaaagg cgagatcacc aaggtagtcg
      181 gcaaataatg tctagctaga aattcgttca agccgacgcc gcttcgcggc gcggcttaac
      241 tcaagcgtta gatgcactaa gcacataatt gctcacagcc aaactatcag gtcaagtctg
      301 cttttattat ttttaagcgt gcataataag ccctacacaa attgggagat atatcatgca
      361 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
      421 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
      481 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
      541 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
      601 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
      661 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
      721 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
      781 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
      841 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
      901 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
      961 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     1021 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     1081 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     1141 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     1201 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg
     1261 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     1321 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     1381 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgg ctccgggagc
     1441 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcaggg tgccttgatg
     1501 tgggcgccgg cggtcgagtg gcgacggcgc ggcttgtccg cgccctggta gattgcctgg
     1561 ccgtaggcca gccatttttg agcggccagc ggccgcgata ggccgacgcg aagcggcggg
     1621 gcgtagggag cgcagcgacc gaagggtagg cgctttttgc agctcttcgg ctgtgcgctg
     1681 gccagacagt tatgcacagg ccaggcgggt tttaagagtt ttaataagtt ttaaagagtt
     1741 ttaggcggaa aaatcgcctt ttttctcttt tatatcagtc acttacatgt gtgaccggtt
     1801 cccaatgtac ggctttgggt tcccaatgta cgggttccgg ttcccaatgt acggctttgg
     1861 gttcccaatg tacgtgctat ccacaggaaa gagagctttt cgaccttttt cccctgctag
     1921 ggcaatttgc cctagcatct gctccgtaca ttaggaaccg gcggatgctt cgccctcgat
     1981 caggttgcgg tagcgcatga ctaggatcgg gccagcctgc cccgcctcct ccttcaaatc
     2041 gtactccggc aggtcatttg acccgatcag cttgcgcacg gtgaaacaga acttcttgaa
     2101 ctctccggcg ctgccactgc gttcgtagat cgtcttgaac aaccatctgg cttctgcctt
     2161 gcctgcggcg cggcgtgcca ggcggtagag aaaacggccg atgccgggat cgatcaaaaa
     2221 gtaatcgggg tgaaccgtca gcacgtccgg gttcttgcct tctgtgatct cgcggtacat
     2281 ccaatcagct agctcgatct cgatgtactc cggccgcccg gtttcgctct ttacgatctt
     2341 gtagcggcta atcaaggctt caccctcgga taccgtcacc aggcggccgt tcttggcctt
     2401 cttcgtacgc tgcatggcaa cgtgcgtggt gtttaaccga atgcaggttt ctaccaggtc
     2461 gtctttctgc tttccgccat cggctcgccg gcagaacttg agtacgtccg caacgtgtgg
     2521 acggaacacg cggccgggct tgtctccctt cccttcccgg tatcggttca tggattcggt
     2581 tagatgggaa accgccatca gtaccaggtc gtaatcccac acactggcca tgccggccgg
     2641 ccctgcggaa acctctacgt gcccgtctgg aagctcgtag cggatcacct cgccagctcg
     2701 tcggtcacgc ttcgacagac ggaaaacggc cacgtccatg atgctgcgac tatcgcgggt
     2761 gcccacgtca tagagcatcg gaacgaaaaa atctggttgc tcgtcgccct tgggcggctt
     2821 cctaatcgac ggcgcaccgg ctgccggcgg ttgccgggat tctttgcgga ttcgatcagc
     2881 ggccgcttgc cacgattcac cggggcgtgc ttctgcctcg atgcgttgcc gctgggcggc
     2941 ctgcgcggcc ttcaacttct ccaccaggtc atcacccagc gccgcgccga tttgtaccgg
     3001 gccggatggt ttgcgaccgc tcacgccgat tcctcgggct tgggggttcc agtgccattg
     3061 cagggccggc agacaaccca gccgcttacg cctggccaac cgcccgttcc tccacacatg
     3121 gggcattcca cggcgtcggt gcctggttgt tcttgatttt ccatgccgcc tcctttagcc
     3181 gctaaaattc atctactcat ttattcattt gctcatttac tctggtagct gcgcgatgta
     3241 ttcagatagc agctcggtaa tggtcttgcc ttggcgtacc gcgtacatct tcagcttggt
     3301 gtgatcctcc gccggcaact gaaagttgac ccgcttcatg gctggcgtgt ctgccagtct
     3361 ggccaacgtt gcagccttgc tgctgcgtgc gctcggacgg ccggcactta gcgtgtttgt
     3421 gcttttgctc attttctctt tacctcatta actcaaatga gttttgattt aatttcagcg
     3481 gccagcgcct ggacctcgcg ggcagcgtcg ccctcgggtt ctgattcaag aacggttgtg
     3541 ccggcggcgg cagtgcctgg gtagctcacg cgctgcgtga tacgggactc aagaatgggc
     3601 agctcgtacc cggccagcgc ctcggcaacc tcaccgccga tgcgcgtgcc tttgatcgcc
     3661 cgcgacacga caaaggccgc ttgtagcctt ccatccgtga cctcaatgcg ctgcttaacc
     3721 agctccacca ggtcggcggt ggcccatatg tcgtaagggc ttggctgcac cggaatcagc
     3781 acgaagtcgg ctgccttgat cgcggacaca gccaagtccg ccgcctgggg cgctccgtcg
     3841 atcactacga agtcgcgccg gccgatggcc ttcacgtcgc ggtcaatcgt cgggcggtcg
     3901 atgccgacaa cggttagcgg ttgatcttcc cgcacggccg cccaatcgcg ggcactgccc
     3961 tggggatcgg aatcgactaa cagaacatcg gccccggcga gttgcagggc gcgggctaga
     4021 tgggttgcga tggtcgtctt gcctgacccg cctttctggt taagtacagc gataaccttc
     4081 atgcgttccc cttgcgtatt tgtttattta ctcatcgcat catatacgca gcgaccgcat
     4141 gacgcaagct gttttactca aatacacatc acctttttag acggcggcgc tcggtttctt
     4201 cagcggccaa gctggccggc caggccgcca gcttggcatc agacaaaccg gccaggattt
     4261 catgcagccg cacggttgat acgtgcgcgg gcggctcgaa cacgtacccg gccgcgatca
     4321 tctccgcctc gatctcttcg gtaatgaaaa acggttcgtc ctggccgtcc tggtgcggtt
     4381 tcatgcttgt tcctcttggc gttcattctc ggcggccgcc agggcgtcgg cctcggtcaa
     4441 tgcgtcctca cggaaggcac cgcgccgcct ggcctcggtg ggcgtcactt cctcgctgcg
     4501 ctcaagtgcg cggtacaggg tcgagcgatg cacgccaagc agtgcagccg cctctttcac
     4561 ggtgcggcct tcctggtcga tcagctcgcg ggcgtgcgcg atctgtgccg gggtgagggt
     4621 agggcggggg ccaaacttca cgcctcgggc cttggcggcc tcgcgcccgc tccgggtgcg
     4681 gtcgatgatt agggaacgct cgaactcggc aatgccggcg aacacggtca acaccatgcg
     4741 gccggccggc gtggtggtgt cggcccacgg ctctgccagg ctacgcaggc ccgcgccggc
     4801 ctcctggatg cgctcggcaa tgtccagtag gtcgcgggtg ctgcgggcca ggcggtctag
     4861 cctggtcact gtcacaacgt cgccagggcg taggtggtca agcatcctgg ccagctccgg
     4921 gcggtcgcgc ctggtgccgg tgatcttctc ggaaaacagc ttggtgcagc cggccgcgtg
     4981 cagttcggcc cgttggttgg tcaagtcctg gtcgtcggtg ctgacgcggg catagcccag
     5041 caggccagcg gcggcgctct tgttcatggc gtaatgtctc cggttctagt cgcaagtatt
     5101 ctactttatg cgactaaaac acgcgacaag aaaacgccag gaaaagggca gggcggcagc
     5161 ctgtcgcgta acttaggact tgtgcgacat gtcgttttca gaagagggct gcactgaacg
     5221 tcagaagccg actgcactat agcagcggag gggttggatc aaagtacttt aaagtacttt
     5281 aaagtacttt aaagtacttt gatcccgagg ggaaccctgt ggttggcatg cacatacaaa
     5341 tggacgaacg gataaacctt ttcacgccct tttaaatatc cgattattct aataaacgct
     5401 cttttctctt aggtttaccc gccaatatat cctgtcaaac actgatagtt taaactgaag
     5461 gcgggaaacg acaatctgat ccaagctcaa gctaagcttc gtctcatcag ctaccttgag
     5521 accgtcgact tatattcccc agaacatcag gttaatggcg tttttgatgt cattttcgcg
     5581 gtggctgaga tcagccactt cttccccgat aacggaaacc ggcacactgg ccatatcggt
     5641 ggtcatcatg cgccagcttt catccccgat atgcaccacc gggtaaagtt cacgggagac
     5701 tttatctgac agcagacgtg cactggccag ggggatcacc atccgtcgcc cgggcgtgtc
     5761 aataatatca ctctgtacat ccacaaacag acgataacgg ctctctcttt tataggtgta
     5821 aaccttaaac tgcatttcac cagcccctgt tctcgtcagc aaaagagccg ttcatttcaa
     5881 taaaccgggc gacctcagcc atcccttcct gattttccgc tttccagcgt tcggcacgca
     5941 gacgacgggc ttcattctgc atggttgtgc ttaccagacc ggagatattg acatcatata
     6001 tgccttgagc aactgatagc tgtcgctgtc aactgtcact gtaatacgct gcttcatagc
     6061 atacctcttt ttgacatact tcgggtatac atatcagtat atattcttat accgcaaaaa
     6121 tcagcgcgca aatacgcata ctgttatctg gcttttagta agccggatcc acgcgtttac
     6181 gccccgccct gccactcatc gcagtactgt tgtaattcat taagcattct gccgacatgg
     6241 aagccatcac aaacggcatg atgaacctga atcgccagcg gcatcagcac cttgtcgcct
     6301 tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga agttgtccat attggccacg
     6361 tttaaatcaa aactggtgaa actcacccag ggattggctg agactaaaaa catattctca
     6421 ataaaccctt tagggaaata ggccaggttt tcaccgtaac acgccacatc ttgcgaatat
     6481 atgtgtagaa actgccggaa atcgtcgtgg tattcactcc agagcgatga aaacgtttca
     6541 gtttgctcat ggaaaacggt gtaacaaggg tgaacactat cccatatcac cagctcaccg
     6601 tctttcattg ccatacggaa ttccggatga gcattcatca ggcgggcaag aatgtgaata
     6661 aaggccggat aaaacttgtg cttatttttc tttacggtct ttaaaaaggc cgtaatatcc
     6721 agctgaacgg tctggttata ggtacattga gcaactgact gaaatgcctc aaaatgttct
     6781 ttacgatgcc attgggatat atcaacggtg gtatatccag tgattttttt ctccatttta
     6841 gcttccttag ctcctgaaaa tctcgacgga tcctaactca aaatccacac attatacgag
     6901 ccggaagcat aaagtgtaaa gcctggggtg cctaatgcgg ccgcggtctc agtattaact
     6961 gcagagacgc tgcaggcatg cgacgtcggg ccctctagag gatccccggg taccgcgaat
     7021 tcaattcggc gttaattcag tacattaaaa acgtccgcaa tgtgttatta agttgtctaa
     7081 gcgtcaattt gtttacacca caatatatcc tgccaccagc cagccaacag ctccccgacc
     7141 ggcagctcgg cacaaaatca ccactcgata caggcagccc atcagtccgg gacggcgtca
     7201 gcgggagagc cgttgtaagg cggcagactt tgctcatgtt accgatgcta ttcggaagaa
     7261 cggcaactaa gctgccgggt ttgaaacacg gatgatctcg cggagggtag catgttgatt
     7321 gtaacgatga cagagcgttg ctgcctgtga tcaattcggg cacgaaccca gtggacataa
     7381 gcctgttcgg ttcgtaagct gtaatgcaag tagcgtatgc gctcacgcaa ctggtccaga
     7441 accttgaccg aacgcagcgg tggtaacggc gcagtggcgg ttttcatggc ttgttatgac
     7501 tgtttttttg gggtacagtc tatgcctcgg gcatccaagc agcaagcgcg ttacgccgtg
     7561 ggtcgatgtt tgatgttatg gagcagcaac gatgttacgc agcagggcag tcgccctaaa
     7621 acaaagttaa acatcatggg ggaagcggtg atcgccgaag tatcgactca actatcagag
     7681 gtagttggcg tcatcgagcg ccatctcgaa ccgacgttgc tggccgtaca tttgtacggc
     7741 tccgcagtgg atggcggcct gaagccacac agtgatattg atttgctggt tacggtgacc
     7801 gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg accttttgga aacttcggct
     7861 tcccctggag agagcgagat tctccgcgct gtagaagtca ccattgttgt gcacgacgac
     7921 atcattccgt ggcgttatcc agctaagcgc gaactgcaat ttggagaatg gcagcgcaat
     7981 gacattcttg caggtatctt cgagccagcc acgatcgaca ttgatctggc tatcttgctg
     8041 acaaaagcaa gagaacatag cgttgccttg gtaggtccag cggcggagga actctttgat
     8101 ccggttcctg aacaggatct atttgaggcg ctaaatgaaa ccttaacgct atggaactcg
     8161 ccgcccgact gggctggcga tgagcgaaat gtagtgctta cgttgtcccg catttggtac
     8221 agcgcagtaa ccggcaaaa
//
