LOCUS       sequence-262716-        6582 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Use when making even level constructs (L2,...) if pCk2 position 
            (position 2) has no construct assigned.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6582)
  AUTHORS   Sauret-Gueto S, Frangedakis E, Silvestri L, Rebmann M, Tomaselli M, 
            Markel K, Delmans M, West A, Patron NJ, Haseloff J
  TITLE     Systematic tools for reprogramming plant gene expression in a simple
            model, Marchantia polymorpha.
  JOURNAL   ACS Synth Biol. 2020 Mar 12. doi: 10.1021/acssynbio.9b00511.
  PUBMED    32163700
REFERENCE   2  (bases 1 to 6582)
  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: "ACS Synth 
            Biol. 2020 Mar 12. doi: 10.1021/acssynbio.9b00511."
COMMENT     Sequence Label: pCk2_spacer
FEATURES             Location/Qualifiers
     source          1..6582
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    433..457
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     misc_feature    705..729
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             2030..2659
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             3088..4161
                     /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"
     rep_origin      4227..4421
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(4657..4679)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4765..4905
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4820..4839
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4920..4937)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5091..5679)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5171..5190)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5767..6582)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(5767..6582)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     6490..6509
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 aacagaactt attatttcct tcctcttttc tacagtattt aaagataccc caagaagcta
       61 attataacaa gacgaactcc aattcactgt tccttgcatt ctaaaacctt aaataccaga
      121 aaacagcttt ttcaaagttg ttttcaaagt tggcgtataa catagtatcg acggagccga
      181 ttttgaaacc gcggtgatca caggcagcaa cgctctgtca tcgttacaat caacatgcta
      241 ccctccgcga gatcatccgt gtttcaaacc cggcagctta gttgccgttc ttccgaatag
      301 catcggtaac atgagcaaag tctgccgcct tacaacggct ctcccgctga cgccgtcccg
      361 gactgatggg ctgcctgtat cgagtggtga ttttgtgccg agctgccggt cggggagctg
      421 ttggctggct ggtggcagga tatattgtgg tgtaaacata acgaattgct cttcagcaag
      481 gagtctgagc aggacaactc gcgtagtgag agttacatgt tcgttgggtt cttccgacac
      541 ggacctgagt tggccaacgt cccacctgag gtctgtgccc cggtgatgag aagtgtgcat
      601 ctcgttcttg cagctcgtca gtactttcag aatcatggcg tgcatggtag aatgaccctt
      661 ataacggact tcgacatggc aatcgctata ctgaagagca attctgacag gatatattgg
      721 cgggtaaacc taagagaaaa gagcgtttat tagaataatc ggatatttaa aagggcgtga
      781 aaaggtttat ccgttcgtcc atttgtatgt gcatgccaac cacagggttc ccctcgggat
      841 caaagtactt tgatccaacc cctccgctgc tatagtgcag tcggcttctg acgttcagtg
      901 cagccgtcat ctgaaaacga catgtcgcac aagtcctaag ttacgcgaca ggctgccgcc
      961 ctgccctttt cctggcgttt tcttgtcgcg tgttttagtc gcataaagta gaatacttgc
     1021 gactagaacc ggagacatta cgccatgaac aagagcgccg ccgctggcct gctgggctat
     1081 gcccgcgtca gcaccgacga ccaggacttg accaaccaac gggccgaact gcacgcggcc
     1141 ggctgcacca agctgttttc cgagaagatc accggcacca ggcgcgaccg cccggagctg
     1201 gccaggatgc ttgaccacct acgccctggc gacgttgtga cagtgaccag gctagaccgc
     1261 ctggcccgca gcacccgcga cctactggac attgccgagc gcatccagga ggccggcgcg
     1321 ggcctgcgta gcctggcaga gccgtgggcc gacaccacca cgccggccgg ccgcatggtg
     1381 ttgaccgtgt tcgccggcat tgccgagttc gagcgttccc taatcatcga ccgcacccgg
     1441 agcgggcgcg aggccgccaa ggcccgaggc gtgaagtttg gcccccgccc taccctcacc
     1501 ccggcacaga tcgcgcacgc ccgcgagctg atcgaccagg aaggccgcac cgtgaaagag
     1561 gcggctgcac tgcttggcgt gcatcgctcg accctgtacc gcgcacttga gcgcagcgag
     1621 gaagtgacgc ccaccgaggc caggcggcgc ggtgccttcc gtgaggacgc attgaccgag
     1681 gccgacgccc tggcggccgc cgagaatgaa cgccaagagg aacaagcatg aaaccgcacc
     1741 aggacggcca ggacgaaccg tttttcatta ccgaagagat cgaggcggag atgatcgcgg
     1801 ccgggtacgt gttcgagccg cccgcgcacc tctcaaccgt gcggctgcat gaaatcctgg
     1861 ccggtttgtc tgatgccaag ctggcggcct ggccggccag cttggccgct gaagaaaccg
     1921 agcgccgccg tctaaaaagg tgatgtgtat ttgagtaaaa cagcttgcgt catgcggtcg
     1981 ctgcgtatat gatgcgatga gtaaataaac aaatacgcaa ggggaacgca tgaaggttat
     2041 cgctgtactt aaccagaaag gcgggtcagg caagacgacc atcgcaaccc atctagcccg
     2101 cgccctgcaa ctcgccgggg ccgatgttct gttagtcgat tccgatcccc agggcagtgc
     2161 ccgcgattgg gcggccgtgc gggaagatca accgctaacc gttgtcggca tcgaccgccc
     2221 gacgattgac cgcgacgtga aggccatcgg ccggcgcgac ttcgtagtga tcgacggagc
     2281 gccccaggcg gcggacttgg ctgtgtccgc gatcaaggca gccgacttcg tgctgattcc
     2341 ggtgcagcca agcccttacg acatatgggc caccgccgac ctggtggagc tggttaagca
     2401 gcgcattgag gtcacggatg gaaggctaca agcggccttt gtcgtgtcgc gggcgatcaa
     2461 aggcacgcgc atcggcggtg aggttgccga ggcgctggcc gggtacgagc tgcccattct
     2521 tgagtcccgt atcacgcagc gcgtgagcta cccaggcact gccgccgccg gcacaaccgt
     2581 tcttgaatca gaacccgagg gcgacgctgc ccgcgaggtc caggcgctgg ccgctgaaat
     2641 taaatcaaaa ctcatttgag ttaatgaggt aaagagaaaa tgagcaaaag cacaaacacg
     2701 ctaagtgccg gccgtccgag cgcacgcagc agcaaggctg caacgttggc cagcctggca
     2761 gacacgccag ccatgaagcg ggtcaacttt cagttgccgg cggaggatca caccaagctg
     2821 aagatgtacg cggtacgcca aggcaagacc attaccgagc tgctatctga atacatcgcg
     2881 cagctaccag agtaaatgag caaatgaata aatgagtaga tgaattttag cggctaaagg
     2941 aggcggcatg gaaaatcaag aacaaccagg caccgacgcc gtggaatgcc ccatgtgtgg
     3001 aggaacgggc ggttggccag gcgtaagcgg ctgggttgcc tgccggccct gcaatggcac
     3061 tggaaccccc aagcccgagg aatcggcgtg agcggtcgca aaccatccgg cccggtacaa
     3121 atcggcgcgg cgctgggtga tgacctggtg gagaagttga aggccgcgca ggccgcccag
     3181 cggcaacgca tcgaggcaga agcacgcccc ggtgaatcgt ggcaagcggc cgctgatcga
     3241 atccgcaaag aatcccggca accgccggca gccggtgcgc cgtcgattag gaagccgccc
     3301 aagggcgacg agcaaccaga ttttttcgtt ccgatgctct atgacgtggg cacccgcgat
     3361 agtcgcagca tcatggacgt ggccgttttc cgtctgtcga agcgtgaccg acgagctggc
     3421 gaggtgatcc gctacgagct tccagacggg cacgtagagg tttccgcagg gccggccggc
     3481 atggccagtg tgtgggatta cgacctggta ctgatggcgg tttcccatct aaccgaatcc
     3541 atgaaccgat accgggaagg gaagggagac aagcccggcc gcgtgttccg tccacacgtt
     3601 gcggacgtac tcaagttctg ccggcgagcc gatggcggaa agcagaaaga cgacctggta
     3661 gaaacctgca ttcggttaaa caccacgcac gttgccatgc agcgtacgaa gaaggccaag
     3721 aacggccgcc tggtgacggt atccgagggt gaagccttga ttagccgcta caagatcgta
     3781 aagagcgaaa ccgggcggcc ggagtacatc gagatcgagc tagctgattg gatgtaccgc
     3841 gagatcacag aaggcaagaa cccggacgtg ctgacggttc accccgatta ctttttgatc
     3901 gatcccggca tcggccgttt tctctaccgc ctggcacgcc gcgccgcagg caaggcagaa
     3961 gccagatggt tgttcaagac gatctacgaa cgcagtggca gcgccggaga gttcaagaag
     4021 ttctgtttca ccgtgcgcaa gctgatcggg tcaaatgacc tgccggagta cgatttgaag
     4081 gaggaggcgg ggcaggctgg cccgatccta gtcatgcgct accgcaacct gatcgagggc
     4141 gaagcatccg ccggttccta atgtacggag cagatgctag ggcaaattgc cctagcaggg
     4201 gaaaaaggtc gaaaaggact ctttcctgtg gatagcacgt acattgggaa cccaaagccg
     4261 tacattggga accggaaccc gtacattggg aacccaaagc cgtacattgg gaaccggtca
     4321 cacatgtaag tgactgatat aaaagagaaa aaaggcgatt tttccgccta aaactcttta
     4381 aaacttatta aaactcttaa aacccgcctg gcctgtgcat aactgtctgg ccagcgcaca
     4441 gccgaagtgc tgcaaaaagc gcctaccctt cggtcgctgc gctccctacg ccccgccgct
     4501 tcgcgtcggc ctatcgcggc cgctggccgc tcaaaaatgg ctggcctacg gccaggcaat
     4561 ctaccagggc gcggacaagc cgcgccgtcg ccactcgacc gccggcgccc acatcaaggc
     4621 accctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccggt
     4681 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     4741 agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt
     4801 gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg
     4861 tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgctc atccgcttcc
     4921 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     4981 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     5041 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     5101 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     5161 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     5221 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     5281 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     5341 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     5401 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     5461 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     5521 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     5581 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     5641 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     5701 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgcattct
     5761 aggtgattag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
     5821 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
     5881 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
     5941 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
     6001 gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac
     6061 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
     6121 tgattgcgcc tgagcgagtc gaaatacgcg atcgctgtta aaaggacaat tacaaacagg
     6181 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
     6241 aggatattct tctaatacct ggaatgctgt tttccctggg atcgcagtgg tgagtaacca
     6301 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     6361 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     6421 cagaaacaac tctggcgcat cgggcttccc atacaatcgg tagattgtcg cacctgattg
     6481 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     6541 tcgcggcctt gagcaagacg tttcccgttg aatatggctc at
//
