LOCUS       sequence-390574-        4445 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor vector to restore L2 destination via BsaI (reverse 
            orientation).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4445)
  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 4445)
  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-Reverse-Donor-BsaI
FEATURES             Location/Qualifiers
     source          1..4445
                     /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(686..691,692..709,710..716)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             770..1450
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     CDS             1770..2075
                     /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     1954..1973
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(2152..2171)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2153..2171)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2477..2495)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2563..2667
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2668..2736,2737..3528)
                     /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(2886..2905)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3699..4287
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4188..4207
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(4441..4445,1..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 gattttggtc tctaccgcac tttttgcagg
      661 tgcggccgca ttaggcaccc caggctttac actttatgct tccggctcgt ataatgtgtg
      721 gattttgagt taggatccgt cgagattttc aggagctaag gaagctaaaa tggagaaaaa
      781 aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac attttgaggc
      841 atttcagtca gtcgctcaat gtacctataa ccagaccgtt cagcttgata ttacggcctt
      901 tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc acattcttgc
      961 ccgcctgatg aatgctcatc cggaattccg tatggcaatg aaagacggtg agctggtgat
     1021 atgggatagt gttcaccctt gttacaccgt tttccatgag caaactgaaa cgttttcatc
     1081 gctctggagt gaataccacg acgatttccg gcagtttcta cacatatatt cgcaagatgt
     1141 ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg tttattgaga atatgttttt
     1201 cgtttcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg ccaatatgga
     1261 caacttcttc gcccccgttt tcaccatggg caaatattat acgcaaggcg acaaggtgct
     1321 gatgccgctg gcgattcagg ttcatcatgc cgtttgtgat ggcttccatg tcggcagaat
     1381 gcttaatgaa ttacaacagt actgcgatga gtggcaggcg gggcgtaatc tagaggatcc
     1441 ggcttactaa aagccagata acagtatgcg tatttgcgcg ctgatttttg cggtataaga
     1501 atatatactg atatgtatac ccgaagtatg tcaaaaggag gtatgctatg aagcagcgta
     1561 ttacagtgac agttgacagc gacagctatc agttgctcaa ggcatatatg atgtcaatat
     1621 ctccggtctg gtaagcacaa ccatgcagaa tgaagcccgt cgtctgcgtg ccgaacgctg
     1681 gaaagcggaa aatcaggaag ggatggctga ggtcgcccgg tttattgaaa tgaacggctc
     1741 ttttgctgac gagaacaggg gctggtgaaa tgcagtttaa ggtttacacc tataaaagag
     1801 agagccgtta tcgtctgttt gtggatgtac agagtgatat tattgacacg cccgggcgac
     1861 ggatggtgat ccccctggcc agtgcacgtc tgctgtcaga taaagtctcc cgtgaacttt
     1921 acccggtggt gcatatcggg gatgaaagct ggcgcatgat gaccaccgat atggccagtg
     1981 tgccggtttc cgttatcggg gaagaagtgg ctgatctcag ccaccgcgaa aatgacatca
     2041 aaaacgccat taacctgatg ttctggggaa tataacacct gcaaaacggt agtgagagac
     2101 caaaatcttg ctgaaaaact cgagccatcc ggaagatctg gcggccgctc tccctatagt
     2161 gagtcgtatt acgccggatg gatatggtgt tcaggcacaa gtgttaaagc agttgatttt
     2221 attcactatg atgaaaaaaa caatgaatgg aacctgctcc aagttaaaaa tagagataat
     2281 accgaaaact catcgagtag taagattaga gataatacaa caataaaaaa atggtttaga
     2341 acttactcac agcgtgatgc tactaattgg gacaattttc cagatgaagt atcatctaag
     2401 aatttaaatg aagaagactt cagagctttt gttaaaaatt atttggcaaa aataatataa
     2461 ttcggctgca ggggcggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     2521 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     2581 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     2641 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     2701 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     2761 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     2821 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     2881 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     2941 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     3001 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     3061 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     3121 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     3181 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     3241 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     3301 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     3361 aaatctggag ccggtgagcg tgggtcacgc ggtatcattg cagcactggg gccagatggt
     3421 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     3481 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     3541 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     3601 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     3661 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     3721 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     3781 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     3841 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     3901 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     3961 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     4021 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     4081 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     4141 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     4201 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     4261 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     4321 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     4381 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     4441 agcga
//
