LOCUS       sequence-274970-        6582 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Use when making even level constructs (L2,...) if pCk4 position 
            (position 4) 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: pCk4_spacer
FEATURES             Location/Qualifiers
     source          1..6582
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(70..87)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(241..829)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(321..340)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(917..1732)
                     /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(917..1732)
                     /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     1640..1659
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     misc_feature    2165..2189
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     misc_feature    2437..2461
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             3762..4391
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             4820..5893
                     /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      5959..6153
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(6389..6411)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    join(6497..6582,1..55)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6552..6571
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgctc
       61 atccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
      121 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
      181 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
      241 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
      301 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
      361 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
      421 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
      481 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
      541 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
      601 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
      661 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
      721 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
      781 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
      841 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
      901 catgcattct aggtgattag aaaaactcat cgagcatcaa atgaaactgc aatttattca
      961 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
     1021 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
     1081 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
     1141 caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga
     1201 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
     1261 tattcattcg tgattgcgcc tgagcgagtc gaaatacgcg atcgctgtta aaaggacaat
     1321 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
     1381 cacctgaatc aggatattct tctaatacct ggaatgctgt tttccctggg atcgcagtgg
     1441 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     1501 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     1561 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcgg tagattgtcg
     1621 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     1681 tggaatttaa tcgcggcctt gagcaagacg tttcccgttg aatatggctc ataacagaac
     1741 ttattatttc cttcctcttt tctacagtat ttaaagatac cccaagaagc taattataac
     1801 aagacgaact ccaattcact gttccttgca ttctaaaacc ttaaatacca gaaaacagct
     1861 ttttcaaagt tgttttcaaa gttggcgtat aacatagtat cgacggagcc gattttgaaa
     1921 ccgcggtgat cacaggcagc aacgctctgt catcgttaca atcaacatgc taccctccgc
     1981 gagatcatcc gtgtttcaaa cccggcagct tagttgccgt tcttccgaat agcatcggta
     2041 acatgagcaa agtctgccgc cttacaacgg ctctcccgct gacgccgtcc cggactgatg
     2101 ggctgcctgt atcgagtggt gattttgtgc cgagctgccg gtcggggagc tgttggctgg
     2161 ctggtggcag gatatattgt ggtgtaaaca taacgaattg ctcttcacag aggagtctga
     2221 gcaggacaac tcgcgtagtg agagttacat gttcgttggg ttcttccgac acggacctga
     2281 gttggccaac gtcccacctg aggtctgtgc cccggtgatg agaagtgtgc atctcgttct
     2341 tgcagctcgt cagtactttc agaatcatgg cgtgcatggt agaatgaccc ttataacgga
     2401 cttcgacatg gcaatcgcta ggttgaagag caattctgac aggatatatt ggcgggtaaa
     2461 cctaagagaa aagagcgttt attagaataa tcggatattt aaaagggcgt gaaaaggttt
     2521 atccgttcgt ccatttgtat gtgcatgcca accacagggt tcccctcggg atcaaagtac
     2581 tttgatccaa cccctccgct gctatagtgc agtcggcttc tgacgttcag tgcagccgtc
     2641 atctgaaaac gacatgtcgc acaagtccta agttacgcga caggctgccg ccctgccctt
     2701 ttcctggcgt tttcttgtcg cgtgttttag tcgcataaag tagaatactt gcgactagaa
     2761 ccggagacat tacgccatga acaagagcgc cgccgctggc ctgctgggct atgcccgcgt
     2821 cagcaccgac gaccaggact tgaccaacca acgggccgaa ctgcacgcgg ccggctgcac
     2881 caagctgttt tccgagaaga tcaccggcac caggcgcgac cgcccggagc tggccaggat
     2941 gcttgaccac ctacgccctg gcgacgttgt gacagtgacc aggctagacc gcctggcccg
     3001 cagcacccgc gacctactgg acattgccga gcgcatccag gaggccggcg cgggcctgcg
     3061 tagcctggca gagccgtggg ccgacaccac cacgccggcc ggccgcatgg tgttgaccgt
     3121 gttcgccggc attgccgagt tcgagcgttc cctaatcatc gaccgcaccc ggagcgggcg
     3181 cgaggccgcc aaggcccgag gcgtgaagtt tggcccccgc cctaccctca ccccggcaca
     3241 gatcgcgcac gcccgcgagc tgatcgacca ggaaggccgc accgtgaaag aggcggctgc
     3301 actgcttggc gtgcatcgct cgaccctgta ccgcgcactt gagcgcagcg aggaagtgac
     3361 gcccaccgag gccaggcggc gcggtgcctt ccgtgaggac gcattgaccg aggccgacgc
     3421 cctggcggcc gccgagaatg aacgccaaga ggaacaagca tgaaaccgca ccaggacggc
     3481 caggacgaac cgtttttcat taccgaagag atcgaggcgg agatgatcgc ggccgggtac
     3541 gtgttcgagc cgcccgcgca cctctcaacc gtgcggctgc atgaaatcct ggccggtttg
     3601 tctgatgcca agctggcggc ctggccggcc agcttggccg ctgaagaaac cgagcgccgc
     3661 cgtctaaaaa ggtgatgtgt atttgagtaa aacagcttgc gtcatgcggt cgctgcgtat
     3721 atgatgcgat gagtaaataa acaaatacgc aaggggaacg catgaaggtt atcgctgtac
     3781 ttaaccagaa aggcgggtca ggcaagacga ccatcgcaac ccatctagcc cgcgccctgc
     3841 aactcgccgg ggccgatgtt ctgttagtcg attccgatcc ccagggcagt gcccgcgatt
     3901 gggcggccgt gcgggaagat caaccgctaa ccgttgtcgg catcgaccgc ccgacgattg
     3961 accgcgacgt gaaggccatc ggccggcgcg acttcgtagt gatcgacgga gcgccccagg
     4021 cggcggactt ggctgtgtcc gcgatcaagg cagccgactt cgtgctgatt ccggtgcagc
     4081 caagccctta cgacatatgg gccaccgccg acctggtgga gctggttaag cagcgcattg
     4141 aggtcacgga tggaaggcta caagcggcct ttgtcgtgtc gcgggcgatc aaaggcacgc
     4201 gcatcggcgg tgaggttgcc gaggcgctgg ccgggtacga gctgcccatt cttgagtccc
     4261 gtatcacgca gcgcgtgagc tacccaggca ctgccgccgc cggcacaacc gttcttgaat
     4321 cagaacccga gggcgacgct gcccgcgagg tccaggcgct ggccgctgaa attaaatcaa
     4381 aactcatttg agttaatgag gtaaagagaa aatgagcaaa agcacaaaca cgctaagtgc
     4441 cggccgtccg agcgcacgca gcagcaaggc tgcaacgttg gccagcctgg cagacacgcc
     4501 agccatgaag cgggtcaact ttcagttgcc ggcggaggat cacaccaagc tgaagatgta
     4561 cgcggtacgc caaggcaaga ccattaccga gctgctatct gaatacatcg cgcagctacc
     4621 agagtaaatg agcaaatgaa taaatgagta gatgaatttt agcggctaaa ggaggcggca
     4681 tggaaaatca agaacaacca ggcaccgacg ccgtggaatg ccccatgtgt ggaggaacgg
     4741 gcggttggcc aggcgtaagc ggctgggttg cctgccggcc ctgcaatggc actggaaccc
     4801 ccaagcccga ggaatcggcg tgagcggtcg caaaccatcc ggcccggtac aaatcggcgc
     4861 ggcgctgggt gatgacctgg tggagaagtt gaaggccgcg caggccgccc agcggcaacg
     4921 catcgaggca gaagcacgcc ccggtgaatc gtggcaagcg gccgctgatc gaatccgcaa
     4981 agaatcccgg caaccgccgg cagccggtgc gccgtcgatt aggaagccgc ccaagggcga
     5041 cgagcaacca gattttttcg ttccgatgct ctatgacgtg ggcacccgcg atagtcgcag
     5101 catcatggac gtggccgttt tccgtctgtc gaagcgtgac cgacgagctg gcgaggtgat
     5161 ccgctacgag cttccagacg ggcacgtaga ggtttccgca gggccggccg gcatggccag
     5221 tgtgtgggat tacgacctgg tactgatggc ggtttcccat ctaaccgaat ccatgaaccg
     5281 ataccgggaa gggaagggag acaagcccgg ccgcgtgttc cgtccacacg ttgcggacgt
     5341 actcaagttc tgccggcgag ccgatggcgg aaagcagaaa gacgacctgg tagaaacctg
     5401 cattcggtta aacaccacgc acgttgccat gcagcgtacg aagaaggcca agaacggccg
     5461 cctggtgacg gtatccgagg gtgaagcctt gattagccgc tacaagatcg taaagagcga
     5521 aaccgggcgg ccggagtaca tcgagatcga gctagctgat tggatgtacc gcgagatcac
     5581 agaaggcaag aacccggacg tgctgacggt tcaccccgat tactttttga tcgatcccgg
     5641 catcggccgt tttctctacc gcctggcacg ccgcgccgca ggcaaggcag aagccagatg
     5701 gttgttcaag acgatctacg aacgcagtgg cagcgccgga gagttcaaga agttctgttt
     5761 caccgtgcgc aagctgatcg ggtcaaatga cctgccggag tacgatttga aggaggaggc
     5821 ggggcaggct ggcccgatcc tagtcatgcg ctaccgcaac ctgatcgagg gcgaagcatc
     5881 cgccggttcc taatgtacgg agcagatgct agggcaaatt gccctagcag gggaaaaagg
     5941 tcgaaaagga ctctttcctg tggatagcac gtacattggg aacccaaagc cgtacattgg
     6001 gaaccggaac ccgtacattg ggaacccaaa gccgtacatt gggaaccggt cacacatgta
     6061 agtgactgat ataaaagaga aaaaaggcga tttttccgcc taaaactctt taaaacttat
     6121 taaaactctt aaaacccgcc tggcctgtgc ataactgtct ggccagcgca cagccgaagt
     6181 gctgcaaaaa gcgcctaccc ttcggtcgct gcgctcccta cgccccgccg cttcgcgtcg
     6241 gcctatcgcg gccgctggcc gctcaaaaat ggctggccta cggccaggca atctaccagg
     6301 gcgcggacaa gccgcgccgt cgccactcga ccgccggcgc ccacatcaag gcaccctgcc
     6361 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gtgacggtca
     6421 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     6481 ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg
     6541 gcttaactat gcggcatcag agcagattgt actgagagtg ca
//
