LOCUS       sequence-447309-        8212 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty spectinomycin 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 8212)
  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 8212)
  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: pVP076
FEATURES             Location/Qualifiers
     source          1..8212
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     430..449
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     683..700
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    715..855
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(781..800)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     941..963
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     rep_origin      1199..1393
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     CDS             complement(1459..2532)
                     /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(2961..3590)
                     /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    4921..4945
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(5025..5330)
                     /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(5127..5146)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(5672..6331)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFLVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6246..6265
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(6385..6391,6392..6409,6410..6415))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    6572..6596
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     CDS             7117..7908
                     /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      join(8154..8212,1..529)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
       61 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      121 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      181 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      241 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      301 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      361 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      421 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      481 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      541 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      601 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      661 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      721 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag
      781 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
      841 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
      901 ctgctcccgg catccgctta cagacaagct gtgaccggct ccgggagctg catgtgtcag
      961 aggttttcac cgtcatcacc gaaacgcgcg aggcagggtg ccttgatgtg ggcgccggcg
     1021 gtcgagtggc gacggcgcgg cttgtccgcg ccctggtaga ttgcctggcc gtaggccagc
     1081 catttttgag cggccagcgg ccgcgatagg ccgacgcgaa gcggcggggc gtagggagcg
     1141 cagcgaccga agggtaggcg ctttttgcag ctcttcggct gtgcgctggc cagacagtta
     1201 tgcacaggcc aggcgggttt taagagtttt aataagtttt aaagagtttt aggcggaaaa
     1261 atcgcctttt ttctctttta tatcagtcac ttacatgtgt gaccggttcc caatgtacgg
     1321 ctttgggttc ccaatgtacg ggttccggtt cccaatgtac ggctttgggt tcccaatgta
     1381 cgtgctatcc acaggaaaga gagcttttcg acctttttcc cctgctaggg caatttgccc
     1441 tagcatctgc tccgtacatt aggaaccggc ggatgcttcg ccctcgatca ggttgcggta
     1501 gcgcatgact aggatcgggc cagcctgccc cgcctcctcc ttcaaatcgt actccggcag
     1561 gtcatttgac ccgatcagct tgcgcacggt gaaacagaac ttcttgaact ctccggcgct
     1621 gccactgcgt tcgtagatcg tcttgaacaa ccatctggct tctgccttgc ctgcggcgcg
     1681 gcgtgccagg cggtagagaa aacggccgat gccgggatcg atcaaaaagt aatcggggtg
     1741 aaccgtcagc acgtccgggt tcttgccttc tgtgatctcg cggtacatcc aatcagctag
     1801 ctcgatctcg atgtactccg gccgcccggt ttcgctcttt acgatcttgt agcggctaat
     1861 caaggcttca ccctcggata ccgtcaccag gcggccgttc ttggccttct tcgtacgctg
     1921 catggcaacg tgcgtggtgt ttaaccgaat gcaggtttct accaggtcgt ctttctgctt
     1981 tccgccatcg gctcgccggc agaacttgag tacgtccgca acgtgtggac ggaacacgcg
     2041 gccgggcttg tctcccttcc cttcccggta tcggttcatg gattcggtta gatgggaaac
     2101 cgccatcagt accaggtcgt aatcccacac actggccatg ccggccggcc ctgcggaaac
     2161 ctctacgtgc ccgtctggaa gctcgtagcg gatcacctcg ccagctcgtc ggtcacgctt
     2221 cgacagacgg aaaacggcca cgtccatgat gctgcgacta tcgcgggtgc ccacgtcata
     2281 gagcatcgga acgaaaaaat ctggttgctc gtcgcccttg ggcggcttcc taatcgacgg
     2341 cgcaccggct gccggcggtt gccgggattc tttgcggatt cgatcagcgg ccgcttgcca
     2401 cgattcaccg gggcgtgctt ctgcctcgat gcgttgccgc tgggcggcct gcgcggcctt
     2461 caacttctcc accaggtcat cacccagcgc cgcgccgatt tgtaccgggc cggatggttt
     2521 gcgaccgctc acgccgattc ctcgggcttg ggggttccag tgccattgca gggccggcag
     2581 acaacccagc cgcttacgcc tggccaaccg cccgttcctc cacacatggg gcattccacg
     2641 gcgtcggtgc ctggttgttc ttgattttcc atgccgcctc ctttagccgc taaaattcat
     2701 ctactcattt attcatttgc tcatttactc tggtagctgc gcgatgtatt cagatagcag
     2761 ctcggtaatg gtcttgcctt ggcgtaccgc gtacatcttc agcttggtgt gatcctccgc
     2821 cggcaactga aagttgaccc gcttcatggc tggcgtgtct gccaggctgg ccaacgttgc
     2881 agccttgctg ctgcgtgcgc tcggacggcc ggcacttagc gtgtttgtgc ttttgctcat
     2941 tttctcttta cctcattaac tcaaatgagt tttgatttaa tttcagcggc cagcgcctgg
     3001 acctcgcggg cagcgtcgcc ctcgggttct gattcaagaa cggttgtgcc ggcggcggca
     3061 gtgcctgggt agctcacgcg ctgcgtgata cgggactcaa gaatgggcag ctcgtacccg
     3121 gccagcgcct cggcaacctc accgccgatg cgcgtgcctt tgatcgcccg cgacacgaca
     3181 aaggccgctt gtagccttcc atccgtgacc tcaatgcgct gcttaaccag ctccaccagg
     3241 tcggcggtgg cccatatgtc gtaagggctt ggctgcaccg gaatcagcac gaagtcggct
     3301 gccttgatcg cggacacagc caagtccgcc gcctggggcg ctccgtcgat cactacgaag
     3361 tcgcgccggc cgatggcctt cacgtcgcgg tcaatcgtcg ggcggtcgat gccgacaacg
     3421 gttagcggtt gatcttcccg cacggccgcc caatcgcggg cactgccctg gggatcggaa
     3481 tcgactaaca gaacatcggc cccggcgagt tgcagggcgc gggctagatg ggttgcgatg
     3541 gtcgtcttgc ctgacccgcc tttctggtta agtacagcga taaccttcat gcgttcccct
     3601 tgcgtatttg tttatttact catcgcatca tatacgcagc gaccgcatga cgcaagctgt
     3661 tttactcaaa tacacatcac ctttttagac ggcggcgctc ggtttcttca gcggccaagc
     3721 tggccggcca ggccgccagc ttggcatcag acaaaccggc caggatttca tgcagccgca
     3781 cggttgatac gtgcgcgggc ggctcgaaca cgtacccggc cgcgatcatc tccgcctcga
     3841 tctcttcggt aatgaaaaac ggttcgtcct ggccgtcctg gtgcggtttc atgcttgttc
     3901 ctcttggcgt tcattctcgg cggccgccag ggcgtcggcc tcggtcaatg cgtcctcacg
     3961 gaaggcaccg cgccgcctgg cctcggtggg cgtcacttcc tcgctgcgct caagtgcgcg
     4021 gtacagggtc gagcgatgca cgccaagcag tgcagccgcc tctttcacgg tgcggccttc
     4081 ctggtcgatc agctcgcggg cgtgcgcgat ctgtgccggg gtgagggtag ggcgggggcc
     4141 aaacttcacg cctcgggcct tggcggcctc gcgcccgctc cgggtgcggt cgatgattag
     4201 ggaacgctcg aactcggcaa tgccggcgaa cacggtcaac accatgcggc cggccggcgt
     4261 ggtggtgtcg gcccacggct ctgccaggct acgcaggccc gcgccggcct cctggatgcg
     4321 ctcggcaatg tccagtaggt cgcgggtgct gcgggccagg cggtctagcc tggtcactgt
     4381 cacaacgtcg ccagggcgta ggtggtcaag catcctggcc agctccgggc ggtcgcgcct
     4441 ggtgccggtg atcttctcgg aaaacagctt ggtgcagccg gccgcgtgca gttcggcccg
     4501 ttggttggtc aagtcctggt cgtcggtgct gacgcgggca tagcccagca ggccagcggc
     4561 ggcgctcttg ttcatggcgt aatgtctccg gttctagtcg caagtattct actttatgcg
     4621 actaaaacac gcgacaagaa aacgccagga aaagggcagg gcggcagcct gtcgcgtaac
     4681 ttaggacttg tgcgacatgt cgttttcaga agagggctgc actgaacgtc agaagccgac
     4741 tgcactatag cagcggaggg gttggatcaa agtactttaa agtactttaa agtactttaa
     4801 agtactttga tcccgagggg aaccctgtgg ttggcatgca catacaaatg gacgaacgga
     4861 taaacctttt cacgcccttt taaatatccg attattctaa taaacgctct tttctcttag
     4921 gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac
     4981 aatctgatcc aagctcaagc taagcttacc ttgagaccgt cgacttatat tccccagaac
     5041 atcaggttaa tggcgttttt gatgtcattt tcgcggtggc tgagatcagc cacttcttcc
     5101 ccgataacgg aaaccggcac actggccata tcggtggtca tcatgcgcca gctttcatcc
     5161 ccgatatgca ccaccgggta aagttcacgg gagactttat ctgacagcag acgtgcactg
     5221 gccaggggga tcaccatccg tcgcccgggc gtgtcaataa tatcactctg tacatccaca
     5281 aacagacgat aacggctctc tcttttatag gtgtaaacct taaactgcat ttcaccagcc
     5341 cctgttctcg tcagcaaaag agccgttcat ttcaataaac cgggcgacct cagccatccc
     5401 ttcctgattt tccgctttcc agcgttcggc acgcagacga cgggcttcat tctgcatggt
     5461 tgtgcttacc agaccggaga tattgacatc atatatgcct tgagcaactg atagctgtcg
     5521 ctgtcaactg tcactgtaat acgctgcttc atagcatacc tctttttgac atacttcggg
     5581 tatacatatc agtatatatt cttataccgc aaaaatcagc gcgcaaatac gcatactgtt
     5641 atctggcttt tagtaagccg gatccacgcg tttacgcccc gccctgccac tcatcgcagt
     5701 actgttgtaa ttcattaagc attctgccga catggaagcc atcacaaacg gcatgatgaa
     5761 cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt ataatatttg cccatggtga
     5821 aaacgggggc gaagaagttg tccatattgg ccacgtttaa atcaaaactg gtgaaactca
     5881 cccagggatt ggctgagact aaaaacatat tctcaataaa ccctttaggg aaataggcca
     5941 ggttttcacc gtaacacgcc acatcttgcg aatatatgtg tagaaactgc cggaaatcgt
     6001 cgtggtattc actccagagc gatgaaaacg tttcagtttg ctcatggaaa acggtgtaac
     6061 aagggtgaac actatcccat atcaccagct caccgtcttt cattgccata cggaattccg
     6121 gatgagcatt catcaggcgg gcaagaatgt gaataaaggc cggataaaac ttgtgcttat
     6181 ttttctttac ggtctttaaa aaggccgtaa tatccagctg aacggtctgg ttataggtac
     6241 attgagcaac tgactgaaat gcctcaaaat gttctttacg atgccattgg gatatatcaa
     6301 cggtggtata tccagtgatt tttttctcca ttttagcttc cttagctcct gaaaatctcg
     6361 acggatccta actcaaaatc cacacattat acgagccgga agcataaagt gtaaagcctg
     6421 gggtgcctaa tgcggccgcg gtctcagtat ctgcaggcat gcgacgtcgg gccctctaga
     6481 ggatccccgg gtaccgcgaa ttcaattcgg cgttaattca gtacattaaa aacgtccgca
     6541 atgtgttatt aagttgtcta agcgtcaatt tgtttacacc acaatatatc ctgccaccag
     6601 ccagccaaca gctccccgac cggcagctcg gcacaaaatc accactcgat acaggcagcc
     6661 catcagtccg ggacggcgtc agcgggagag ccgttgtaag gcggcagact ttgctcatgt
     6721 taccgatgct attcggaaga acggcaacta agctgccggg tttgaaacac ggatgatctc
     6781 gcggagggta gcatgttgat tgtaacgatg acagagcgtt gctgcctgtg atcaattcgg
     6841 gcacgaaccc agtggacata agcctgttcg gttcgtaagc tgtaatgcaa gtagcgtatg
     6901 cgctcacgca actggtccag aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg
     6961 gttttcatgg cttgttatga ctgttttttt ggggtacagt ctatgcctcg ggcatccaag
     7021 cagcaagcgc gttacgccgt gggtcgatgt ttgatgttat ggagcagcaa cgatgttacg
     7081 cagcagggca gtcgccctaa aacaaagtta aacatcatgg gggaagcggt gatcgccgaa
     7141 gtatcgactc aactatcaga ggtagttggc gtcatcgagc gccatctcga accgacgttg
     7201 ctggccgtac atttgtacgg ctccgcagtg gatggcggcc tgaagccaca cagtgatatt
     7261 gatttgctgg ttacggtgac cgtaaggctt gatgaaacaa cgcggcgagc tttgatcaac
     7321 gaccttttgg aaacttcggc ttcccctgga gagagcgaga ttctccgcgc tgtagaagtc
     7381 accattgttg tgcacgacga catcattccg tggcgttatc cagctaagcg cgaactgcaa
     7441 tttggagaat ggcagcgcaa tgacattctt gcaggtatct tcgagccagc cacgatcgac
     7501 attgatctgg ctatcttgct gacaaaagca agagaacata gcgttgcctt ggtaggtcca
     7561 gcggcggagg aactctttga tccggttcct gaacaggatc tatttgaggc gctaaatgaa
     7621 accttaacgc tatggaactc gccgcccgac tgggctggcg atgagcgaaa tgtagtgctt
     7681 acgttgtccc gcatttggta cagcgcagta accggcaaaa tcgcgccgaa ggatgtcgct
     7741 gccgactggg caatggagcg cctgccggcc cagtatcagc ccgtcatact tgaagctaga
     7801 caggcttatc ttggacaaga agaagatcgc ttggcctcgc gcgcagatca gttggaagaa
     7861 tttgtccact acgtgaaagg cgagatcacc aaggtagtcg gcaaataatg tctagctaga
     7921 aattcgttca agccgacgcc gcttcgcggc gcggcttaac tcaagcgtta gatgcactaa
     7981 gcacataatt gctcacagcc aaactatcag gtcaagtctg cttttattat ttttaagcgt
     8041 gcataataag ccctacacaa attgggagat atatcatgca tgaccaaaat cccttaacgt
     8101 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     8161 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa accaccgcta cc
//
