LOCUS       sequence-440979-        8386 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MultiGreen 2.0 DE level 1 acceptor vector, kanamycin selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8386)
  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 8386)
  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: pVP314
FEATURES             Location/Qualifiers
     source          1..8386
                     /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(5056..5361)
                     /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(5158..5177)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(5703..6362)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFLVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6277..6296
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(6416..6422,6423..6440,6441..6446))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    6617..6641
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     primer_bind     complement(6923..6942)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6934..7749
                     /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             6934..7749
                     /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(7007..7026)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(8345..8386,1..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 catcagctac
     5041 cttgagaccg tcgacttata ttccccagaa catcaggtta atggcgtttt tgatgtcatt
     5101 ttcgcggtgg ctgagatcag ccacttcttc cccgataacg gaaaccggca cactggccat
     5161 atcggtggtc atcatgcgcc agctttcatc cccgatatgc accaccgggt aaagttcacg
     5221 ggagacttta tctgacagca gacgtgcact ggccaggggg atcaccatcc gtcgcccggg
     5281 cgtgtcaata atatcactct gtacatccac aaacagacga taacggctct ctcttttata
     5341 ggtgtaaacc ttaaactgca tttcaccagc ccctgttctc gtcagcaaaa gagccgttca
     5401 tttcaataaa ccgggcgacc tcagccatcc cttcctgatt ttccgctttc cagcgttcgg
     5461 cacgcagacg acgggcttca ttctgcatgg ttgtgcttac cagaccggag atattgacat
     5521 catatatgcc ttgagcaact gatagctgtc gctgtcaact gtcactgtaa tacgctgctt
     5581 catagcatac ctctttttga catacttcgg gtatacatat cagtatatat tcttataccg
     5641 caaaaatcag cgcgcaaata cgcatactgt tatctggctt ttagtaagcc ggatccacgc
     5701 gtttacgccc cgccctgcca ctcatcgcag tactgttgta attcattaag cattctgccg
     5761 acatggaagc catcacaaac ggcatgatga acctgaatcg ccagcggcat cagcaccttg
     5821 tcgccttgcg tataatattt gcccatggtg aaaacggggg cgaagaagtt gtccatattg
     5881 gccacgttta aatcaaaact ggtgaaactc acccagggat tggctgagac taaaaacata
     5941 ttctcaataa accctttagg gaaataggcc aggttttcac cgtaacacgc cacatcttgc
     6001 gaatatatgt gtagaaactg ccggaaatcg tcgtggtatt cactccagag cgatgaaaac
     6061 gtttcagttt gctcatggaa aacggtgtaa caagggtgaa cactatccca tatcaccagc
     6121 tcaccgtctt tcattgccat acggaattcc ggatgagcat tcatcaggcg ggcaagaatg
     6181 tgaataaagg ccggataaaa cttgtgctta tttttcttta cggtctttaa aaaggccgta
     6241 atatccagct gaacggtctg gttataggta cattgagcaa ctgactgaaa tgcctcaaaa
     6301 tgttctttac gatgccattg ggatatatca acggtggtat atccagtgat ttttttctcc
     6361 attttagctt ccttagctcc tgaaaatctc gacggatcct aactcaaaat ccacacatta
     6421 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgcggccgc ggtctcagta
     6481 ttaactgcag agacgctgca ggcatgcgac gtcgggccct ctagaggatc cccgggtacc
     6541 gcgaattcaa ttcggcgtta attcagtaca ttaaaaacgt ccgcaatgtg ttattaagtt
     6601 gtctaagcgt caatttgttt acaccacaat atatcctgcc accagccagc caacagctcc
     6661 ccgaccggca gctcggcaca aaatcaccac tcgatacagg cagcccatca gtccgggacg
     6721 gcgtcagcgg gagagccgtt gtaaggcggc agactttgct catgttaccg atgctattcg
     6781 gaagaacggc aactaagctg ccgggtttga aacacggatg atctcgcgga gggtagcatg
     6841 ttgattgtaa cgatgacaga gcgttgctgc ctgtgatcca gatcatgaac aataaaactg
     6901 tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtct
     6961 tgctctaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct
     7021 cgcgataatg tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg
     7081 ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg
     7141 gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt
     7201 actcctgatg atgcatggtt actcaccact gcgatccccg ggaaaacagc attccaggta
     7261 ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc
     7321 cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcggctc
     7381 gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag
     7441 cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca
     7501 ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg
     7561 aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt
     7621 gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa
     7681 aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag
     7741 tttttctaac agcggcggag gaactctttg atccggttcc tgaacaggat ctatttgagg
     7801 cgctaaatga aaccttaacg ctatggaact cgccgcccga ctgggctggc gatgagcgaa
     7861 atgtagtgct tacgttgtcc cgcatttggt acagcgcagt aaccggcaaa atcgcgccga
     7921 aggatgtcgc tgccgactgg gcaatggagc gcctgccggc ccagtatcag cccgtcatac
     7981 ttgaagctag acaggcttat cttggacaag aagaagatcg cttggcctcg cgcgcagatc
     8041 agttggaaga atttgtccac tacgtgaaag gcgagatcac caaggtagtc ggcaaataat
     8101 gtctagctag aaattcgttc aagccgacgc cgcttcgcgg cgcggcttaa ctcaagcgtt
     8161 agatgcacta agcacataat tgctcacagc caaactatca ggtcaagtct gcttttatta
     8221 tttttaagcg tgcataataa gccctacaca aattgggaga tatatcatgc atgaccaaaa
     8281 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     8341 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaac
//
