LOCUS       sequence-274967-        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"
     primer_bind     375..394
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     628..645
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    660..800
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(726..745)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     886..908
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     rep_origin      1144..1338
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     CDS             complement(1404..2477)
                     /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"
     CDS             complement(2906..3535)
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     misc_feature    4836..4860
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     misc_feature    5108..5132
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     CDS             5565..6380
                     /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             5565..6380
                     /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     complement(5638..5657)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6468..6582,1..474)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
       61 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
      121 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
      181 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
      241 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
      301 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
      361 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
      421 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
      481 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
      541 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
      601 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggatgagcgc
      661 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact
      721 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
      781 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
      841 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtcaccggg agctgcatgt
      901 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gggtgccttg atgtgggcgc
      961 cggcggtcga gtggcgacgg cgcggcttgt ccgcgccctg gtagattgcc tggccgtagg
     1021 ccagccattt ttgagcggcc agcggccgcg ataggccgac gcgaagcggc ggggcgtagg
     1081 gagcgcagcg accgaagggt aggcgctttt tgcagcactt cggctgtgcg ctggccagac
     1141 agttatgcac aggccaggcg ggttttaaga gttttaataa gttttaaaga gttttaggcg
     1201 gaaaaatcgc cttttttctc ttttatatca gtcacttaca tgtgtgaccg gttcccaatg
     1261 tacggctttg ggttcccaat gtacgggttc cggttcccaa tgtacggctt tgggttccca
     1321 atgtacgtgc tatccacagg aaagagtcct tttcgacctt tttcccctgc tagggcaatt
     1381 tgccctagca tctgctccgt acattaggaa ccggcggatg cttcgccctc gatcaggttg
     1441 cggtagcgca tgactaggat cgggccagcc tgccccgcct cctccttcaa atcgtactcc
     1501 ggcaggtcat ttgacccgat cagcttgcgc acggtgaaac agaacttctt gaactctccg
     1561 gcgctgccac tgcgttcgta gatcgtcttg aacaaccatc tggcttctgc cttgcctgcg
     1621 gcgcggcgtg ccaggcggta gagaaaacgg ccgatgccgg gatcgatcaa aaagtaatcg
     1681 gggtgaaccg tcagcacgtc cgggttcttg ccttctgtga tctcgcggta catccaatca
     1741 gctagctcga tctcgatgta ctccggccgc ccggtttcgc tctttacgat cttgtagcgg
     1801 ctaatcaagg cttcaccctc ggataccgtc accaggcggc cgttcttggc cttcttcgta
     1861 cgctgcatgg caacgtgcgt ggtgtttaac cgaatgcagg tttctaccag gtcgtctttc
     1921 tgctttccgc catcggctcg ccggcagaac ttgagtacgt ccgcaacgtg tggacggaac
     1981 acgcggccgg gcttgtctcc cttcccttcc cggtatcggt tcatggattc ggttagatgg
     2041 gaaaccgcca tcagtaccag gtcgtaatcc cacacactgg ccatgccggc cggccctgcg
     2101 gaaacctcta cgtgcccgtc tggaagctcg tagcggatca cctcgccagc tcgtcggtca
     2161 cgcttcgaca gacggaaaac ggccacgtcc atgatgctgc gactatcgcg ggtgcccacg
     2221 tcatagagca tcggaacgaa aaaatctggt tgctcgtcgc ccttgggcgg cttcctaatc
     2281 gacggcgcac cggctgccgg cggttgccgg gattctttgc ggattcgatc agcggccgct
     2341 tgccacgatt caccggggcg tgcttctgcc tcgatgcgtt gccgctgggc ggcctgcgcg
     2401 gccttcaact tctccaccag gtcatcaccc agcgccgcgc cgatttgtac cgggccggat
     2461 ggtttgcgac cgctcacgcc gattcctcgg gcttgggggt tccagtgcca ttgcagggcc
     2521 ggcaggcaac ccagccgctt acgcctggcc aaccgcccgt tcctccacac atggggcatt
     2581 ccacggcgtc ggtgcctggt tgttcttgat tttccatgcc gcctccttta gccgctaaaa
     2641 ttcatctact catttattca tttgctcatt tactctggta gctgcgcgat gtattcagat
     2701 agcagctcgg taatggtctt gccttggcgt accgcgtaca tcttcagctt ggtgtgatcc
     2761 tccgccggca actgaaagtt gacccgcttc atggctggcg tgtctgccag gctggccaac
     2821 gttgcagcct tgctgctgcg tgcgctcgga cggccggcac ttagcgtgtt tgtgcttttg
     2881 ctcattttct ctttacctca ttaactcaaa tgagttttga tttaatttca gcggccagcg
     2941 cctggacctc gcgggcagcg tcgccctcgg gttctgattc aagaacggtt gtgccggcgg
     3001 cggcagtgcc tgggtagctc acgcgctgcg tgatacggga ctcaagaatg ggcagctcgt
     3061 acccggccag cgcctcggca acctcaccgc cgatgcgcgt gcctttgatc gcccgcgaca
     3121 cgacaaaggc cgcttgtagc cttccatccg tgacctcaat gcgctgctta accagctcca
     3181 ccaggtcggc ggtggcccat atgtcgtaag ggcttggctg caccggaatc agcacgaagt
     3241 cggctgcctt gatcgcggac acagccaagt ccgccgcctg gggcgctccg tcgatcacta
     3301 cgaagtcgcg ccggccgatg gccttcacgt cgcggtcaat cgtcgggcgg tcgatgccga
     3361 caacggttag cggttgatct tcccgcacgg ccgcccaatc gcgggcactg ccctggggat
     3421 cggaatcgac taacagaaca tcggccccgg cgagttgcag ggcgcgggct agatgggttg
     3481 cgatggtcgt cttgcctgac ccgcctttct ggttaagtac agcgataacc ttcatgcgtt
     3541 ccccttgcgt atttgtttat ttactcatcg catcatatac gcagcgaccg catgacgcaa
     3601 gctgttttac tcaaatacac atcacctttt tagacggcgg cgctcggttt cttcagcggc
     3661 caagctggcc ggccaggccg ccagcttggc atcagacaaa ccggccagga tttcatgcag
     3721 ccgcacggtt gagaggtgcg cgggcggctc gaacacgtac ccggccgcga tcatctccgc
     3781 ctcgatctct tcggtaatga aaaacggttc gtcctggccg tcctggtgcg gtttcatgct
     3841 tgttcctctt ggcgttcatt ctcggcggcc gccagggcgt cggcctcggt caatgcgtcc
     3901 tcacggaagg caccgcgccg cctggcctcg gtgggcgtca cttcctcgct gcgctcaagt
     3961 gcgcggtaca gggtcgagcg atgcacgcca agcagtgcag ccgcctcttt cacggtgcgg
     4021 ccttcctggt cgatcagctc gcgggcgtgc gcgatctgtg ccggggtgag ggtagggcgg
     4081 gggccaaact tcacgcctcg ggccttggcg gcctcgcgcc cgctccgggt gcggtcgatg
     4141 attagggaac gctcgaactc ggcaatgccg gcgaacacgg tcaacaccat gcggccggcc
     4201 ggcgtggtgg tgtcggccca cggctctgcc aggctacgca ggcccgcgcc ggcctcctgg
     4261 atgcgctcgg caatgtccag taggtcgcgg gtgctgcggg ccaggcggtc tagcctggtc
     4321 actgtcacaa cgtcgccagg gcgtaggtgg tcaagcatcc tggccagctc cgggcggtcg
     4381 cgcctggtgc cggtgatctt ctcggaaaac agcttggtgc agccggccgc gtgcagttcg
     4441 gcccgttggt tggtcaagtc ctggtcgtcg gtgctgacgc gggcatagcc cagcaggcca
     4501 gcggcggcgc tcttgttcat ggcgtaatgt ctccggttct agtcgcaagt attctacttt
     4561 atgcgactaa aacacgcgac aagaaaacgc caggaaaagg gcagggcggc agcctgtcgc
     4621 gtaacttagg acttgtgcga catgtcgttt tcagatgacg gctgcactga acgtcagaag
     4681 ccgactgcac tatagcagcg gaggggttgg atcaaagtac tttgatcccg aggggaaccc
     4741 tgtggttggc atgcacatac aaatggacga acggataaac cttttcacgc ccttttaaat
     4801 atccgattat tctaataaac gctcttttct cttaggttta cccgccaata tatcctgtca
     4861 gaattgctct tcagtatagc gattgccatg tcgaagtccg ttataagggt cattctacca
     4921 tgcacgccat gattctgaaa gtactgacga gctgcaagaa cgagatgcac acttctcatc
     4981 accggggcac agacctcagg tgggacgttg gccaactcag gtccgtgtcg gaagaaccca
     5041 acgaacatgt aactctcact acgcgagttg tcctgctcag actccttgct gaagagcaat
     5101 tcgttatgtt tacaccacaa tatatcctgc caccagccag ccaacagctc cccgaccggc
     5161 agctcggcac aaaatcacca ctcgatacag gcagcccatc agtccgggac ggcgtcagcg
     5221 ggagagccgt tgtaaggcgg cagactttgc tcatgttacc gatgctattc ggaagaacgg
     5281 caactaagct gccgggtttg aaacacggat gatctcgcgg agggtagcat gttgattgta
     5341 acgatgacag agcgttgctg cctgtgatca ccgcggtttc aaaatcggct ccgtcgatac
     5401 tatgttatac gccaactttg aaaacaactt tgaaaaagct gttttctggt atttaaggtt
     5461 ttagaatgca aggaacagtg aattggagtt cgtcttgtta taattagctt cttggggtat
     5521 ctttaaatac tgtagaaaag aggaaggaaa taataagttc tgttatgagc catattcaac
     5581 gggaaacgtc ttgctcaagg ccgcgattaa attccaacat ggatgctgat ttatatgggt
     5641 ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctaccga ttgtatggga
     5701 agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta
     5761 cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc
     5821 attttatccg tactcctgat gatgcatggt tactcaccac tgcgatccca gggaaaacag
     5881 cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag
     5941 tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg
     6001 tatttcgact cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt
     6061 ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataaacttt
     6121 tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt
     6181 ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat
     6241 accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac
     6301 ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga
     6361 tgctcgatga gtttttctaa tcacctagaa tgcatgacca aaatccctta acgtgagttt
     6421 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     6481 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     6541 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ct
//
