LOCUS       sequence-438343-        3749 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GreenGate EF base entry vector with an Esp3I domesticated backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3749)
  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 3749)
  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: pVP299
FEATURES             Location/Qualifiers
     source          1..3749
                     /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"
     protein_bind    835..856
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(871..876,877..894,895..901)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    909..925
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        939..957
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     939..956
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             1018..1677
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1084..1103)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1696..2001
                     /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     1880..1899
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(2062..2081)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2063..2081)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2091..2113)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2307..2326)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2486..2504)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2572..2676
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2677..2745,2746..3537)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2895..2914)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(3708..3749,1..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 actgttcttc 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 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
      781 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
      841 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
      901 gtgtggaatt gtgagcggat aacaatttca cacgagctat ttaggtgaca ctatagaagt
      961 gaagcttggt ctcactgcaa ggatccagtg ctaacatggt ctagaaggag gtcagctatg
     1021 gagaaaaaaa tcactggata taccaccgtt gatatatccc aatggcatcg taaagaacat
     1081 tttgaggcat ttcagtcagt tgctcaatgt acctataacc agaccgttca gctggatatt
     1141 acggcctttt taaagaccgt aaagaaaaat aagcacaagt tttatccggc ctttattcac
     1201 attcttgccc gcctgatgaa tgctcatccg gagttccgta tggcaatgaa agacggtgag
     1261 ctggtgatat gggatagtgt tcacccttgt tacaccgttt tccatgagca aactgaaacg
     1321 ttttcatcgc tctggagtga ataccacgac gatttccggc agtttctaca catatattcg
     1381 caagatgtgg cgtgttacgg tgaaaacctg gcctatttcc ctaaagggtt tattgagaat
     1441 atgtttttcg tctcagccaa tccctgggtg agtttcacca gttttgattt aaacgtggcc
     1501 aatatggaca acttcttcgc ccccgttttc accatgggca aatattatac gcaaggcgac
     1561 aaggtgctga tgccgctggc gattcaggtt catcatgccg tttgtgatgg cttccatgtc
     1621 ggcagaatgc ttaatgaatt acaacagtac tgcgatgagt ggcagggcgg ggcgtaagag
     1681 ctcaggagga cagctatgca gtttaaggtt tacacctata aaagagagag ccgttatcgt
     1741 ctgtttgtgg atgtacagag tgatattatt gacacgcccg ggcgacggat ggtgatcccc
     1801 ctggccagtg cacgtctgct gtcagataaa gtctcccgtg aactttaccc agtggtgcat
     1861 atcggggatg aaagctggcg catgatgacc accgatatgg ccagtgtgcc agtctccgtt
     1921 atcggggaag aagtggctga tctcagccac cgcgaaaatg acatcaaaaa cgccattaac
     1981 ctgatgttct ggggaatata actgcagagg aggtaatcaa tgccacaaca ctggtggtac
     2041 cactatgaga ccgaattctc gccctatagt gagtcgtatt acctggccgt cgttttacaa
     2101 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     2161 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     2221 agcctgaatg gcgaatggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     2281 tcacaccgca tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     2341 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     2401 gcttacagac aagctgtgac cgtcttcggg agctgcatgt gtcagaggtt ttcaccgtca
     2461 tcaccgaaac gcgctagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     2521 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     2581 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     2641 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     2701 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     2761 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     2821 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     2881 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     2941 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     3001 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     3061 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     3121 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     3181 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     3241 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     3301 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     3361 attgctgata aatctggagc cggtgagcgt ggctctcgag gtatcattgc agcactgggg
     3421 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     3481 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     3541 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     3601 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     3661 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     3721 tttctgcgcg taatctgctg cttgcaaac
//
