LOCUS       sequence-390567-        4437 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor vector to restore L2 destination via BsaI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4437)
  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 4437)
  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-BsaI
FEATURES             Location/Qualifiers
     source          1..4437
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     68..86
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     392..411
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        392..410
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        join(507..512,513..530,531..537)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             591..1271
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     CDS             1591..1896
                     /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     1775..1794
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     promoter        complement(join(2356..2362,2363..2380,2381..2386))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    2401..2422
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2542..2559)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2713..3301)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2793..2812)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3472..4263,4264..4332))
                     /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     4095..4114
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4333..4437)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
       61 ctataaaaat aggcgtatca cgaggccgcc cctgcagccg aattatatta tttttgccaa
      121 ataattttta acaaaagctc tgaagtcttc ttcatttaaa ttcttagatg atacttcatc
      181 tggaaaattg tcccaattag tagcatcacg ctgtgagtaa gttctaaacc atttttttat
      241 tgttgtatta tctctaatct tactactcga tgagttttcg gtattatctc tatttttaac
      301 ttggagcagg ttccattcat tgtttttttc atcatagtga ataaaatcaa ctgctttaac
      361 acttgtgcct gaacaccata tccatccggc gtaatacgac tcactatagg gagagcggcc
      421 gccagatctt ccggatggct cgagtttttc agcaagattt tggtctctac cgttttgcag
      481 gtgcggccgc attaggcacc ccaggcttta cactttatgc ttccggctcg tataatgtgt
      541 ggattttgag ttaggatccg tcgagatttt caggagctaa ggaagctaaa atggagaaaa
      601 aaatcactgg atataccacc gttgatatat cccaatggca tcgtaaagaa cattttgagg
      661 catttcagtc agtcgctcaa tgtacctata accagaccgt tcagctggat attacggcct
      721 ttttaaagac cgtaaagaaa aataagcaca agttttatcc ggcctttatt cacattcttg
      781 cccgcctgat gaatgctcat ccggaattcc gtatggcaat gaaagacggt gagctggtga
      841 tatgggatag tgttcaccct tgttacaccg ttttccatga gcaaactgaa acgttttcat
      901 cgctctggag tgaataccac gacgatttcc ggcagtttct acacatatat tcgcaagatg
      961 tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg gtttattgag aatatgtttt
     1021 tcgtttcagc caatccctgg gtgagtttca ccagttttga tttaaacgtg gccaatatgg
     1081 acaacttctt cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc
     1141 tgatgccgct ggcgattcag gttcatcatg ccgtttgtga tggcttccat gtcggcagaa
     1201 tgcttaatga attacaacag tactgcgatg agtggcaggc ggggcgtaat ctagaggatc
     1261 cggcttacta aaagccagat aacagtatgc gtatttgcgc gctgattttt gcggtataag
     1321 aatatatact gatatgtata cccgaagtat gtcaaaagga ggtatgctat gaagcagcgt
     1381 attacagtga cagttgacag cgacagctat cagttgctca aggcatatat gatgtcaata
     1441 tctccggtct ggtaagcaca accatgcaga atgaagcccg tcgtctgcgt gccgaacgct
     1501 ggaaagcgga aaatcaggaa gggatggctg aggtcgcccg gtttattgaa atgaacggct
     1561 cttttgctga cgagaacagg ggctggtgaa atgcagttta aggtttacac ctataaaaga
     1621 gagagccgtt atcgtctgtt tgtggatgta cagagtgata ttattgacac gcccgggcga
     1681 cggatggtga tccccctggc cagtgcacgt ctgctgtcag ataaagtctc ccgtgaactt
     1741 tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga tgaccaccga tatggccagt
     1801 gtgccggttt ccgttatcgg ggaagaagtg gctgatctca gccaccgcga aaatgacatc
     1861 aaaaacgcca ttaacctgat gttctgggga atataacacc tgcaaaaagt gagagaccaa
     1921 aatctttcta gaagatctcc tacaatattc tcagctgcca tggaaaatcg atgttcttct
     1981 tttattctct caagattttc aggctgtata ttaaaactta tattaagaac tatgctaacc
     2041 acctcatcag gaaccgttgt aggtggcgtg ggttttcttg gcaatcgact ctcatgaaaa
     2101 ctacgagcta aatattcaat atgttcctct tgaccaactt tattctgcat tttttttgaa
     2161 cgaggtttag agcaagcttc aggaaactga gacaggaatt ttattaaaaa tttaaatttt
     2221 gaagaaagtt cagggttaat agcatccatt ttttgctttg caagttcctc agcattctta
     2281 acaaaagatg tctcttttga catgtttaaa gtttaaacct cctgtgtgaa attattatcc
     2341 gctcataatt ccacacatta tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     2401 atgagtgagc taactcacat taattgcgtt gcgctcactg ccaattgctt tccagtcggg
     2461 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     2521 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     2581 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     2641 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     2701 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     2761 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     2821 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     2881 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     2941 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     3001 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     3061 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     3121 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     3181 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     3241 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     3301 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     3361 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     3421 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     3481 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     3541 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     3601 aatgataccg cgtgacccac gctcaccggc tccagattta tcagcaataa accagccagc
     3661 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     3721 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     3781 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     3841 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     3901 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     3961 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     4021 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     4081 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     4141 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     4201 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     4261 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     4321 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     4381 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcg
//
