LOCUS       sequence-274968-        6582 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Use when making even level constructs (L2,...) if pCk3 position 
            (position 3) 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: pCk3_spacer
FEATURES             Location/Qualifiers
     source          1..6582
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(54..71)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(225..813)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(305..324)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(901..1716)
                     /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(901..1716)
                     /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     1624..1643
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     misc_feature    2149..2173
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     misc_feature    2421..2445
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             3746..4375
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             4804..5877
                     /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      5943..6137
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(6373..6395)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    join(6481..6582,1..39)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6536..6555
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcatccgc ttcctcgctc
       61 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
      121 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
      181 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
      241 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
      301 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
      361 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
      421 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
      481 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
      541 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
      601 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
      661 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
      721 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
      781 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
      841 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgca ttctaggtga
      901 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
      961 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
     1021 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
     1081 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
     1141 tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca
     1201 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
     1261 cgcctgagcg agtcgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
     1321 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     1381 ttcttctaat acctggaatg ctgttttccc tgggatcgca gtggtgagta accatgcatc
     1441 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     1501 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     1561 caactctggc gcatcgggct tcccatacaa tcggtagatt gtcgcacctg attgcccgac
     1621 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     1681 ccttgagcaa gacgtttccc gttgaatatg gctcataaca gaacttatta tttccttcct
     1741 cttttctaca gtatttaaag ataccccaag aagctaatta taacaagacg aactccaatt
     1801 cactgttcct tgcattctaa aaccttaaat accagaaaac agctttttca aagttgtttt
     1861 caaagttggc gtataacata gtatcgacgg agccgatttt gaaaccgcgg tgatcacagg
     1921 cagcaacgct ctgtcatcgt tacaatcaac atgctaccct ccgcgagatc atccgtgttt
     1981 caaacccggc agcttagttg ccgttcttcc gaatagcatc ggtaacatga gcaaagtctg
     2041 ccgccttaca acggctctcc cgctgacgcc gtcccggact gatgggctgc ctgtatcgag
     2101 tggtgatttt gtgccgagct gccggtcggg gagctgttgg ctggctggtg gcaggatata
     2161 ttgtggtgta aacataacga attgctcttc atacaggagt ctgagcagga caactcgcgt
     2221 agtgagagtt acatgttcgt tgggttcttc cgacacggac ctgagttggc caacgtccca
     2281 cctgaggtct gtgccccggt gatgagaagt gtgcatctcg ttcttgcagc tcgtcagtac
     2341 tttcagaatc atggcgtgca tggtagaatg acccttataa cggacttcga catggcaatc
     2401 gctacagtga agagcaattc tgacaggata tattggcggg taaacctaag agaaaagagc
     2461 gtttattaga ataatcggat atttaaaagg gcgtgaaaag gtttatccgt tcgtccattt
     2521 gtatgtgcat gccaaccaca gggttcccct cgggatcaaa gtactttgat ccaacccctc
     2581 cgctgctata gtgcagtcgg cttctgacgt tcagtgcagc cgtcatctga aaacgacatg
     2641 tcgcacaagt cctaagttac gcgacaggct gccgccctgc ccttttcctg gcgttttctt
     2701 gtcgcgtgtt ttagtcgcat aaagtagaat acttgcgact agaaccggag acattacgcc
     2761 atgaacaaga gcgccgccgc tggcctgctg ggctatgccc gcgtcagcac cgacgaccag
     2821 gacttgacca accaacgggc cgaactgcac gcggccggct gcaccaagct gttttccgag
     2881 aagatcaccg gcaccaggcg cgaccgcccg gagctggcca ggatgcttga ccacctacgc
     2941 cctggcgacg ttgtgacagt gaccaggcta gaccgcctgg cccgcagcac ccgcgaccta
     3001 ctggacattg ccgagcgcat ccaggaggcc ggcgcgggcc tgcgtagcct ggcagagccg
     3061 tgggccgaca ccaccacgcc ggccggccgc atggtgttga ccgtgttcgc cggcattgcc
     3121 gagttcgagc gttccctaat catcgaccgc acccggagcg ggcgcgaggc cgccaaggcc
     3181 cgaggcgtga agtttggccc ccgccctacc ctcaccccgg cacagatcgc gcacgcccgc
     3241 gagctgatcg accaggaagg ccgcaccgtg aaagaggcgg ctgcactgct tggcgtgcat
     3301 cgctcgaccc tgtaccgcgc acttgagcgc agcgaggaag tgacgcccac cgaggccagg
     3361 cggcgcggtg ccttccgtga ggacgcattg accgaggccg acgccctggc ggccgccgag
     3421 aatgaacgcc aagaggaaca agcatgaaac cgcaccagga cggccaggac gaaccgtttt
     3481 tcattaccga agagatcgag gcggagatga tcgcggccgg gtacgtgttc gagccgcccg
     3541 cgcacctctc aaccgtgcgg ctgcatgaaa tcctggccgg tttgtctgat gccaagctgg
     3601 cggcctggcc ggccagcttg gccgctgaag aaaccgagcg ccgccgtcta aaaaggtgat
     3661 gtgtatttga gtaaaacagc ttgcgtcatg cggtcgctgc gtatatgatg cgatgagtaa
     3721 ataaacaaat acgcaagggg aacgcatgaa ggttatcgct gtacttaacc agaaaggcgg
     3781 gtcaggcaag acgaccatcg caacccatct agcccgcgcc ctgcaactcg ccggggccga
     3841 tgttctgtta gtcgattccg atccccaggg cagtgcccgc gattgggcgg ccgtgcggga
     3901 agatcaaccg ctaaccgttg tcggcatcga ccgcccgacg attgaccgcg acgtgaaggc
     3961 catcggccgg cgcgacttcg tagtgatcga cggagcgccc caggcggcgg acttggctgt
     4021 gtccgcgatc aaggcagccg acttcgtgct gattccggtg cagccaagcc cttacgacat
     4081 atgggccacc gccgacctgg tggagctggt taagcagcgc attgaggtca cggatggaag
     4141 gctacaagcg gcctttgtcg tgtcgcgggc gatcaaaggc acgcgcatcg gcggtgaggt
     4201 tgccgaggcg ctggccgggt acgagctgcc cattcttgag tcccgtatca cgcagcgcgt
     4261 gagctaccca ggcactgccg ccgccggcac aaccgttctt gaatcagaac ccgagggcga
     4321 cgctgcccgc gaggtccagg cgctggccgc tgaaattaaa tcaaaactca tttgagttaa
     4381 tgaggtaaag agaaaatgag caaaagcaca aacacgctaa gtgccggccg tccgagcgca
     4441 cgcagcagca aggctgcaac gttggccagc ctggcagaca cgccagccat gaagcgggtc
     4501 aactttcagt tgccggcgga ggatcacacc aagctgaaga tgtacgcggt acgccaaggc
     4561 aagaccatta ccgagctgct atctgaatac atcgcgcagc taccagagta aatgagcaaa
     4621 tgaataaatg agtagatgaa ttttagcggc taaaggaggc ggcatggaaa atcaagaaca
     4681 accaggcacc gacgccgtgg aatgccccat gtgtggagga acgggcggtt ggccaggcgt
     4741 aagcggctgg gttgcctgcc ggccctgcaa tggcactgga acccccaagc ccgaggaatc
     4801 ggcgtgagcg gtcgcaaacc atccggcccg gtacaaatcg gcgcggcgct gggtgatgac
     4861 ctggtggaga agttgaaggc cgcgcaggcc gcccagcggc aacgcatcga ggcagaagca
     4921 cgccccggtg aatcgtggca agcggccgct gatcgaatcc gcaaagaatc ccggcaaccg
     4981 ccggcagccg gtgcgccgtc gattaggaag ccgcccaagg gcgacgagca accagatttt
     5041 ttcgttccga tgctctatga cgtgggcacc cgcgatagtc gcagcatcat ggacgtggcc
     5101 gttttccgtc tgtcgaagcg tgaccgacga gctggcgagg tgatccgcta cgagcttcca
     5161 gacgggcacg tagaggtttc cgcagggccg gccggcatgg ccagtgtgtg ggattacgac
     5221 ctggtactga tggcggtttc ccatctaacc gaatccatga accgataccg ggaagggaag
     5281 ggagacaagc ccggccgcgt gttccgtcca cacgttgcgg acgtactcaa gttctgccgg
     5341 cgagccgatg gcggaaagca gaaagacgac ctggtagaaa cctgcattcg gttaaacacc
     5401 acgcacgttg ccatgcagcg tacgaagaag gccaagaacg gccgcctggt gacggtatcc
     5461 gagggtgaag ccttgattag ccgctacaag atcgtaaaga gcgaaaccgg gcggccggag
     5521 tacatcgaga tcgagctagc tgattggatg taccgcgaga tcacagaagg caagaacccg
     5581 gacgtgctga cggttcaccc cgattacttt ttgatcgatc ccggcatcgg ccgttttctc
     5641 taccgcctgg cacgccgcgc cgcaggcaag gcagaagcca gatggttgtt caagacgatc
     5701 tacgaacgca gtggcagcgc cggagagttc aagaagttct gtttcaccgt gcgcaagctg
     5761 atcgggtcaa atgacctgcc ggagtacgat ttgaaggagg aggcggggca ggctggcccg
     5821 atcctagtca tgcgctaccg caacctgatc gagggcgaag catccgccgg ttcctaatgt
     5881 acggagcaga tgctagggca aattgcccta gcaggggaaa aaggtcgaaa aggactcttt
     5941 cctgtggata gcacgtacat tgggaaccca aagccgtaca ttgggaaccg gaacccgtac
     6001 attgggaacc caaagccgta cattgggaac cggtcacaca tgtaagtgac tgatataaaa
     6061 gagaaaaaag gcgatttttc cgcctaaaac tctttaaaac ttattaaaac tcttaaaacc
     6121 cgcctggcct gtgcataact gtctggccag cgcacagccg aagtgctgca aaaagcgcct
     6181 acccttcggt cgctgcgctc cctacgcccc gccgcttcgc gtcggcctat cgcggccgct
     6241 ggccgctcaa aaatggctgg cctacggcca ggcaatctac cagggcgcgg acaagccgcg
     6301 ccgtcgccac tcgaccgccg gcgcccacat caaggcaccc tgcctcgcgc gtttcggtga
     6361 tgacggtgaa aacctctgac acatgcagct cccggtgacg gtcacagctt gtctgtaagc
     6421 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
     6481 cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca
     6541 tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aa
//
