LOCUS       sequence-440971-        8234 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MultiGreen 2.0 BC level 1 acceptor vector, spectinomycin selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8234)
  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 8234)
  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: pVP079
FEATURES             Location/Qualifiers
     source          1..8234
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    119..143
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(234..539)
                     /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(336..355)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(881..1540)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFLVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1455..1474
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(1594..1600,1601..1618,1619..1624))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    1792..1816
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     CDS             2337..3128
                     /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"
     rep_origin      3374..3961
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3862..3881
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4115..4132
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4147..4287
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4213..4232)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4373..4395
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     rep_origin      4631..4825
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     CDS             complement(4891..5964)
                     /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(6393..7022)
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
ORIGIN
        1 tactttgatc ccgaggggaa ccctgtggtt ggcatgcaca tacaaatgga cgaacggata
       61 aaccttttca cgccctttta aatatccgat tattctaata aacgctcttt tctcttaggt
      121 ttacccgcca atatatcctg tcaaacactg atagtttaaa ctgaaggcgg gaaacgacaa
      181 tctgatccaa gctcaagcta agcttcgtct caaacaacct tgagaccgtc gacttatatt
      241 ccccagaaca tcaggttaat ggcgtttttg atgtcatttt cgcggtggct gagatcagcc
      301 acttcttccc cgataacgga aaccggcaca ctggccatat cggtggtcat catgcgccag
      361 ctttcatccc cgatatgcac caccgggtaa agttcacggg agactttatc tgacagcaga
      421 cgtgcactgg ccagggggat caccatccgt cgcccgggcg tgtcaataat atcactctgt
      481 acatccacaa acagacgata acggctctct cttttatagg tgtaaacctt aaactgcatt
      541 tcaccagccc ctgttctcgt cagcaaaaga gccgttcatt tcaataaacc gggcgacctc
      601 agccatccct tcctgatttt ccgctttcca gcgttcggca cgcagacgac gggcttcatt
      661 ctgcatggtt gtgcttacca gaccggagat attgacatca tatatgcctt gagcaactga
      721 tagctgtcgc tgtcaactgt cactgtaata cgctgcttca tagcatacct ctttttgaca
      781 tacttcgggt atacatatca gtatatattc ttataccgca aaaatcagcg cgcaaatacg
      841 catactgtta tctggctttt agtaagccgg atccacgcgt ttacgccccg ccctgccact
      901 catcgcagta ctgttgtaat tcattaagca ttctgccgac atggaagcca tcacaaacgg
      961 catgatgaac ctgaatcgcc agcggcatca gcaccttgtc gccttgcgta taatatttgc
     1021 ccatggtgaa aacgggggcg aagaagttgt ccatattggc cacgtttaaa tcaaaactgg
     1081 tgaaactcac ccagggattg gctgagacta aaaacatatt ctcaataaac cctttaggga
     1141 aataggccag gttttcaccg taacacgcca catcttgcga atatatgtgt agaaactgcc
     1201 ggaaatcgtc gtggtattca ctccagagcg atgaaaacgt ttcagtttgc tcatggaaaa
     1261 cggtgtaaca agggtgaaca ctatcccata tcaccagctc accgtctttc attgccatac
     1321 ggaattccgg atgagcattc atcaggcggg caagaatgtg aataaaggcc ggataaaact
     1381 tgtgcttatt tttctttacg gtctttaaaa aggccgtaat atccagctga acggtctggt
     1441 tataggtaca ttgagcaact gactgaaatg cctcaaaatg ttctttacga tgccattggg
     1501 atatatcaac ggtggtatat ccagtgattt ttttctccat tttagcttcc ttagctcctg
     1561 aaaatctcga cggatcctaa ctcaaaatcc acacattata cgagccggaa gcataaagtg
     1621 taaagcctgg ggtgcctaat gcggccgcgg tctcagtatg gctagagacg ctgcaggcat
     1681 gcgacgtcgg gccctctaga ggatccccgg gtaccgcgaa ttcaattcgg cgttaattca
     1741 gtacattaaa aacgtccgca atgtgttatt aagttgtcta agcgtcaatt tgtttacacc
     1801 acaatatatc ctgccaccag ccagccaaca gctccccgac cggcagctcg gcacaaaatc
     1861 accactcgat acaggcagcc catcagtccg ggacggcgtc agcgggagag ccgttgtaag
     1921 gcggcagact ttgctcatgt taccgatgct attcggaaga acggcaacta agctgccggg
     1981 tttgaaacac ggatgatctc gcggagggta gcatgttgat tgtaacgatg acagagcgtt
     2041 gctgcctgtg atcaattcgg gcacgaaccc agtggacata agcctgttcg gttcgtaagc
     2101 tgtaatgcaa gtagcgtatg cgctcacgca actggtccag aaccttgacc gaacgcagcg
     2161 gtggtaacgg cgcagtggcg gttttcatgg cttgttatga ctgttttttt ggggtacagt
     2221 ctatgcctcg ggcatccaag cagcaagcgc gttacgccgt gggtcgatgt ttgatgttat
     2281 ggagcagcaa cgatgttacg cagcagggca gtcgccctaa aacaaagtta aacatcatgg
     2341 gggaagcggt gatcgccgaa gtatcgactc aactatcaga ggtagttggc gtcatcgagc
     2401 gccatctcga accgacgttg ctggccgtac atttgtacgg ctccgcagtg gatggcggcc
     2461 tgaagccaca cagtgatatt gatttgctgg ttacggtgac cgtaaggctt gatgaaacaa
     2521 cgcggcgagc tttgatcaac gaccttttgg aaacttcggc ttcccctgga gagagcgaga
     2581 ttctccgcgc tgtagaagtc accattgttg tgcacgacga catcattccg tggcgttatc
     2641 cagctaagcg cgaactgcaa tttggagaat ggcagcgcaa tgacattctt gcaggtatct
     2701 tcgagccagc cacgatcgac attgatctgg ctatcttgct gacaaaagca agagaacata
     2761 gcgttgcctt ggtaggtcca gcggcggagg aactctttga tccggttcct gaacaggatc
     2821 tatttgaggc gctaaatgaa accttaacgc tatggaactc gccgcccgac tgggctggcg
     2881 atgagcgaaa tgtagtgctt acgttgtccc gcatttggta cagcgcagta accggcaaaa
     2941 tcgcgccgaa ggatgtcgct gccgactggg caatggagcg cctgccggcc cagtatcagc
     3001 ccgtcatact tgaagctaga caggcttatc ttggacaaga agaagatcgc ttggcctcgc
     3061 gcgcagatca gttggaagaa tttgtccact acgtgaaagg cgagatcacc aaggtagtcg
     3121 gcaaataatg tctagctaga aattcgttca agccgacgcc gcttcgcggc gcggcttaac
     3181 tcaagcgtta gatgcactaa gcacataatt gctcacagcc aaactatcag gtcaagtctg
     3241 cttttattat ttttaagcgt gcataataag ccctacacaa attgggagat atatcatgca
     3301 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     3361 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     3421 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     3481 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     3541 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     3601 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     3661 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     3721 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     3781 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     3841 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     3901 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     3961 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     4021 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     4081 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     4141 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg
     4201 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     4261 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     4321 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgg ctccgggagc
     4381 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcaggg tgccttgatg
     4441 tgggcgccgg cggtcgagtg gcgacggcgc ggcttgtccg cgccctggta gattgcctgg
     4501 ccgtaggcca gccatttttg agcggccagc ggccgcgata ggccgacgcg aagcggcggg
     4561 gcgtagggag cgcagcgacc gaagggtagg cgctttttgc agctcttcgg ctgtgcgctg
     4621 gccagacagt tatgcacagg ccaggcgggt tttaagagtt ttaataagtt ttaaagagtt
     4681 ttaggcggaa aaatcgcctt ttttctcttt tatatcagtc acttacatgt gtgaccggtt
     4741 cccaatgtac ggctttgggt tcccaatgta cgggttccgg ttcccaatgt acggctttgg
     4801 gttcccaatg tacgtgctat ccacaggaaa gagagctttt cgaccttttt cccctgctag
     4861 ggcaatttgc cctagcatct gctccgtaca ttaggaaccg gcggatgctt cgccctcgat
     4921 caggttgcgg tagcgcatga ctaggatcgg gccagcctgc cccgcctcct ccttcaaatc
     4981 gtactccggc aggtcatttg acccgatcag cttgcgcacg gtgaaacaga acttcttgaa
     5041 ctctccggcg ctgccactgc gttcgtagat cgtcttgaac aaccatctgg cttctgcctt
     5101 gcctgcggcg cggcgtgcca ggcggtagag aaaacggccg atgccgggat cgatcaaaaa
     5161 gtaatcgggg tgaaccgtca gcacgtccgg gttcttgcct tctgtgatct cgcggtacat
     5221 ccaatcagct agctcgatct cgatgtactc cggccgcccg gtttcgctct ttacgatctt
     5281 gtagcggcta atcaaggctt caccctcgga taccgtcacc aggcggccgt tcttggcctt
     5341 cttcgtacgc tgcatggcaa cgtgcgtggt gtttaaccga atgcaggttt ctaccaggtc
     5401 gtctttctgc tttccgccat cggctcgccg gcagaacttg agtacgtccg caacgtgtgg
     5461 acggaacacg cggccgggct tgtctccctt cccttcccgg tatcggttca tggattcggt
     5521 tagatgggaa accgccatca gtaccaggtc gtaatcccac acactggcca tgccggccgg
     5581 ccctgcggaa acctctacgt gcccgtctgg aagctcgtag cggatcacct cgccagctcg
     5641 tcggtcacgc ttcgacagac ggaaaacggc cacgtccatg atgctgcgac tatcgcgggt
     5701 gcccacgtca tagagcatcg gaacgaaaaa atctggttgc tcgtcgccct tgggcggctt
     5761 cctaatcgac ggcgcaccgg ctgccggcgg ttgccgggat tctttgcgga ttcgatcagc
     5821 ggccgcttgc cacgattcac cggggcgtgc ttctgcctcg atgcgttgcc gctgggcggc
     5881 ctgcgcggcc ttcaacttct ccaccaggtc atcacccagc gccgcgccga tttgtaccgg
     5941 gccggatggt ttgcgaccgc tcacgccgat tcctcgggct tgggggttcc agtgccattg
     6001 cagggccggc agacaaccca gccgcttacg cctggccaac cgcccgttcc tccacacatg
     6061 gggcattcca cggcgtcggt gcctggttgt tcttgatttt ccatgccgcc tcctttagcc
     6121 gctaaaattc atctactcat ttattcattt gctcatttac tctggtagct gcgcgatgta
     6181 ttcagatagc agctcggtaa tggtcttgcc ttggcgtacc gcgtacatct tcagcttggt
     6241 gtgatcctcc gccggcaact gaaagttgac ccgcttcatg gctggcgtgt ctgccagtct
     6301 ggccaacgtt gcagccttgc tgctgcgtgc gctcggacgg ccggcactta gcgtgtttgt
     6361 gcttttgctc attttctctt tacctcatta actcaaatga gttttgattt aatttcagcg
     6421 gccagcgcct ggacctcgcg ggcagcgtcg ccctcgggtt ctgattcaag aacggttgtg
     6481 ccggcggcgg cagtgcctgg gtagctcacg cgctgcgtga tacgggactc aagaatgggc
     6541 agctcgtacc cggccagcgc ctcggcaacc tcaccgccga tgcgcgtgcc tttgatcgcc
     6601 cgcgacacga caaaggccgc ttgtagcctt ccatccgtga cctcaatgcg ctgcttaacc
     6661 agctccacca ggtcggcggt ggcccatatg tcgtaagggc ttggctgcac cggaatcagc
     6721 acgaagtcgg ctgccttgat cgcggacaca gccaagtccg ccgcctgggg cgctccgtcg
     6781 atcactacga agtcgcgccg gccgatggcc ttcacgtcgc ggtcaatcgt cgggcggtcg
     6841 atgccgacaa cggttagcgg ttgatcttcc cgcacggccg cccaatcgcg ggcactgccc
     6901 tggggatcgg aatcgactaa cagaacatcg gccccggcga gttgcagggc gcgggctaga
     6961 tgggttgcga tggtcgtctt gcctgacccg cctttctggt taagtacagc gataaccttc
     7021 atgcgttccc cttgcgtatt tgtttattta ctcatcgcat catatacgca gcgaccgcat
     7081 gacgcaagct gttttactca aatacacatc acctttttag acggcggcgc tcggtttctt
     7141 cagcggccaa gctggccggc caggccgcca gcttggcatc agacaaaccg gccaggattt
     7201 catgcagccg cacggttgat acgtgcgcgg gcggctcgaa cacgtacccg gccgcgatca
     7261 tctccgcctc gatctcttcg gtaatgaaaa acggttcgtc ctggccgtcc tggtgcggtt
     7321 tcatgcttgt tcctcttggc gttcattctc ggcggccgcc agggcgtcgg cctcggtcaa
     7381 tgcgtcctca cggaaggcac cgcgccgcct ggcctcggtg ggcgtcactt cctcgctgcg
     7441 ctcaagtgcg cggtacaggg tcgagcgatg cacgccaagc agtgcagccg cctctttcac
     7501 ggtgcggcct tcctggtcga tcagctcgcg ggcgtgcgcg atctgtgccg gggtgagggt
     7561 agggcggggg ccaaacttca cgcctcgggc cttggcggcc tcgcgcccgc tccgggtgcg
     7621 gtcgatgatt agggaacgct cgaactcggc aatgccggcg aacacggtca acaccatgcg
     7681 gccggccggc gtggtggtgt cggcccacgg ctctgccagg ctacgcaggc ccgcgccggc
     7741 ctcctggatg cgctcggcaa tgtccagtag gtcgcgggtg ctgcgggcca ggcggtctag
     7801 cctggtcact gtcacaacgt cgccagggcg taggtggtca agcatcctgg ccagctccgg
     7861 gcggtcgcgc ctggtgccgg tgatcttctc ggaaaacagc ttggtgcagc cggccgcgtg
     7921 cagttcggcc cgttggttgg tcaagtcctg gtcgtcggtg ctgacgcggg catagcccag
     7981 caggccagcg gcggcgctct tgttcatggc gtaatgtctc cggttctagt cgcaagtatt
     8041 ctactttatg cgactaaaac acgcgacaag aaaacgccag gaaaagggca gggcggcagc
     8101 ctgtcgcgta acttaggact tgtgcgacat gtcgttttca gaagagggct gcactgaacg
     8161 tcagaagccg actgcactat agcagcggag gggttggatc aaagtacttt aaagtacttt
     8221 aaagtacttt aaag
//
