LOCUS       sequence-390571-        4432 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor vector to restore L2 destination via Esp3I.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4432)
  AUTHORS   Chamness JC, Kumar J, Cruz AJ, Rhuby E, Holum MJ, Cody JP, Tibebu R,
            Gamo ME, Starker CG, Zhang F, Voytas DF
  TITLE     An extensible vector toolkit and parts library for advanced 
            engineering of plant genomes.
  JOURNAL   Plant Genome. 2023 Mar 9:e20312. doi: 10.1002/tpg2.20312.
  PUBMED    36896468
REFERENCE   2  (bases 1 to 4432)
  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: "Plant 
            Genome. 2023 Mar 9:e20312. doi: 10.1002/tpg2.20312."
COMMENT     Sequence Label: pJMC-L2-Donor-Esp3I
FEATURES             Location/Qualifiers
     source          1..4432
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    133..154
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(169..174,175..192,193..199)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     promoter        join(680..685,686..703,704..710)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             764..1444
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     CDS             1764..2069
                     /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     1948..1967
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(2139..2158)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2140..2158)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2464..2482)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2550..2654
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2655..2723,2724..3515)
                     /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(2873..2892)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3686..4274
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4175..4194
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(4428..4432,1..13)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
       61 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcaa ttggcagtga
      121 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
      181 gcttccggct cgtataatgt gtggaattat gagcggataa taatttcaca caggaggttt
      241 aaactttaaa catgtcaaaa gagacatctt ttgttaagaa tgctgaggaa cttgcaaagc
      301 aaaaaatgga tgctattaac cctgaacttt cttcaaaatt taaattttta ataaaattcc
      361 tgtctcagtt tcctgaagct tgctctaaac ctcgttcaaa aaaaatgcag aataaagttg
      421 gtcaagagga acatattgaa tatttagctc gtagttttca tgagagtcga ttgccaagaa
      481 aacccacgcc acctacaacg gttcctgatg aggtggttag catagttctt aatataagtt
      541 ttaatataca gcctgaaaat cttgagagaa taaaagaaga acatcgattt tccatggcag
      601 ctgagaatat tgtaggagat cttctagaaa gattcgtctc taccgttttg caggtgcggc
      661 cgcattaggc accccaggct ttacacttta tgcttccggc tcgtataatg tgtggatttt
      721 gagttaggat ccgtcgagat tttcaggagc taaggaagct aaaatggaga aaaaaatcac
      781 tggatatacc accgttgata tatcccaatg gcatcgtaaa gaacattttg aggcatttca
      841 gtcagtcgct caatgtacct ataaccagac cgttcagctt gatattacgg cctttttaaa
      901 gaccgtaaag aaaaataagc acaagtttta tccggccttt attcacattc ttgcccgcct
      961 gatgaatgct catccggaat tccgtatggc aatgaaagac ggtgagctgg tgatatggga
     1021 tagtgttcac ccttgttaca ccgttttcca tgagcaaact gaaacgtttt catcgctctg
     1081 gagtgaatac cacgacgatt tccggcagtt tctacacata tattcgcaag atgtggcgtg
     1141 ttacggtgaa aacctggcct atttccctaa agggtttatt gagaatatgt ttttcgtttc
     1201 agccaatccc tgggtgagtt tcaccagttt tgatttaaac gtggccaata tggacaactt
     1261 cttcgccccc gttttcacca tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc
     1321 gctggcgatt caggttcatc atgccgtttg tgatggcttc catgtcggca gaatgcttaa
     1381 tgaattacaa cagtactgcg atgagtggca ggcggggcgt aatctagagg atccggctta
     1441 ctaaaagcca gataacagta tgcgtatttg cgcgctgatt tttgcggtat aagaatatat
     1501 actgatatgt atacccgaag tatgtcaaaa ggaggtatgc tatgaagcag cgtattacag
     1561 tgacagttga cagcgacagc tatcagttgc tcaaggcata tatgatgtca atatctccgg
     1621 tctggtaagc acaaccatgc agaatgaagc ccgtcgtctg cgtgccgaac gctggaaagc
     1681 ggaaaatcag gaagggatgg ctgaggtcgc ccggtttatt gaaatgaacg gctcttttgc
     1741 tgacgagaac aggggctggt gaaatgcagt ttaaggttta cacctataaa agagagagcc
     1801 gttatcgtct gtttgtggat gtacagagtg atattattga cacgcccggg cgacggatgg
     1861 tgatccccct ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa ctttacccgg
     1921 tggtgcatat cggggatgaa agctggcgca tgatgaccac cgatatggcc agtgtgccgg
     1981 tttccgttat cggggaagaa gtggctgatc tcagccaccg cgaaaatgac atcaaaaacg
     2041 ccattaacct gatgttctgg ggaatataac acctgcaaaa agtgagagac gatcttgctg
     2101 aaaaactcga gccatccgga agatctggcg gccgctctcc ctatagtgag tcgtattacg
     2161 ccggatggat atggtgttca ggcacaagtg ttaaagcagt tgattttatt cactatgatg
     2221 aaaaaaacaa tgaatggaac ctgctccaag ttaaaaatag agataatacc gaaaactcat
     2281 cgagtagtaa gattagagat aatacaacaa taaaaaaatg gtttagaact tactcacagc
     2341 gtgatgctac taattgggac aattttccag atgaagtatc atctaagaat ttaaatgaag
     2401 aagacttcag agcttttgtt aaaaattatt tggcaaaaat aatataattc ggctgcaggg
     2461 gcggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     2521 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     2581 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     2641 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     2701 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     2761 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     2821 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     2881 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     2941 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     3001 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     3061 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     3121 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     3181 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     3241 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     3301 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     3361 gtgagcgtgg gtcacgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     3421 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     3481 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     3541 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     3601 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     3661 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     3721 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     3781 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     3841 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     3901 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     3961 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     4021 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     4081 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     4141 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     4201 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     4261 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     4321 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     4381 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc ga
//
