LOCUS       sequence-258482-        6106 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For insulated expression in Synechocystis 6803. As pCK306 (rhaBAD 
            rhamnose-inducible promoter, YFP, an E. coli-Synechocystis shuttle 
            vector, chromosomal-integration sites) + terminator ECK120015170.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6106)
  AUTHORS   Kelly CL, Taylor GM, Satkute A, Dekker L, Heap JT
  TITLE     Transcriptional Terminators Allow Leak-Free Chromosomal Integration 
            of Genetic Constructs in Cyanobacteria.
  JOURNAL   Microorganisms. 2019 Aug 16;7(8). pii: microorganisms7080263. doi: 
            10.3390/microorganisms7080263.
  PUBMED    31426276
REFERENCE   2  (bases 1 to 6106)
  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: 
            "Microorganisms."; date: "2019-08-16"; volume: "7(8). pii: 
            microorganisms7080263. doi"; pages: " 10.3390/microorganisms7080263"
COMMENT     Sequence Label: pCK353
FEATURES             Location/Qualifiers
     source          1..6106
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(106..125)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             117..932
                     /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             117..932
                     /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(190..209)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             967..1803
                     /codon_start=1
                     /gene="E. coli rhaS"
                     /product="positive regulator of the rhaB promoter
                     "
                     /label=rhaS
                     /note="activated by L-rhamnose"
                     /translation="MTVLHSVDFFPSGNASVAIEPRLPQADFPEHHHDFHEIVIVEHGT
                     GIHVFNGQPYTITGGTVCFVRDHDRHLYEHTDNLCLTNVLYRSPDRFQFLAGLNQLLPQ
                     ELDGQYPSHWRVNHSVLQQVRQLVAQMEQQEGENDLPSTASREILFMQLLLLLRKSSLQ
                     ENLENSASRLNLLLAWLEDHFADEVNWDAVADQFSLSLRTLHRQLKQQTGLTPQRYLNR
                     LRLMKARHLLRHSEASVTDIAYRCGFSDSNHFSTLFRREFNWSPRDIRQGRDGFLQ"
     rep_origin      3175..3720
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     3621..3640
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3837..3854
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5230..5347
                     /label=rhaB promoter
                     /note="promoter of the E. coli rhaBAD operon, conferring 
                     tight induction with L-rhamnose and repression with 
                     D-glucose in the presence of RhaR and RhaS (Giacalone et 
                     al., 2006)"
     CDS             join(5387..5389,5390..5392,5393..6106)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5432..5453)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5693..5712)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6040..6061
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 ggatccaaag ccacgttgtg tctcaaaatc tctgatgtta cattgcacaa gataaaaata
       61 tatcatcatg aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga
      121 gccatattca acgggaaacg tcttgctcga ggccgcgatt aaattccaac atggatgctg
      181 atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc
      241 gattgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg
      301 ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc
      361 cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc
      421 ccgggaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg
      481 atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta
      541 acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg
      601 atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa
      661 tgcataagct tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg
      721 ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa
      781 tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt
      841 cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc
      901 agtttcattt gatgctcgat gagtttttct aatatttcgc cgtgttgacg acatcaggag
      961 gccagtatga ccgtattaca tagtgtggat ttttttccgt ctggtaacgc gtccgtggcg
     1021 atagaacccc ggctcccgca ggcggatttt cctgaacatc atcatgattt tcatgaaatt
     1081 gtgattgtcg aacatggcac gggtattcat gtgtttaatg ggcagcccta taccatcacc
     1141 ggtggcacgg tctgtttcgt acgcgatcat gatcggcatc tgtatgaaca taccgataat
     1201 ctgtgtctga ccaatgtgct gtatcgctcg ccggatcgat ttcagtttct cgccgggctg
     1261 aatcagttgc tgccacaaga gctggatggg cagtatccgt ctcactggcg cgttaaccac
     1321 agcgtattgc agcaggtgcg acagctggtt gcacagatgg aacagcagga aggggaaaat
     1381 gatttaccct cgaccgccag tcgcgagatc ttgtttatgc aattactgct cttgctgcgt
     1441 aaaagcagtt tgcaggagaa cctggaaaac agcgcatcac gtctcaactt gcttctggcc
     1501 tggctggagg accattttgc cgatgaggtg aattgggatg ccgtggcgga tcaattttct
     1561 ctttcactgc gtacgctaca tcggcagctt aagcagcaaa cgggactgac gcctcagcga
     1621 tacctgaacc gcctgcgact gatgaaagcc cgacatctgc tacgccacag cgaggccagc
     1681 gttactgaca tcgcctatcg ctgtggattc agcgacagta accacttttc gacgcttttt
     1741 cgccgagagt ttaactggtc accgcgtgat attcgccagg gacgggatgg ctttctgcaa
     1801 taatcttggg ggaaattaag accaaactcg atgacctcca aaaagatgta acttctctta
     1861 agatcgatat ggcaacggtg aaaaccgagt tatctgcggt caggatggag ataggtacag
     1921 tcaaggatga tgttaaagat gtcaaagggc gggctaatgc tcaaatttgg gcgttgattc
     1981 ttgccgtcat cggagccata attaccacct tggtgcgttt tggcattttc cctaatccct
     2041 aacaaaaaag cgaccaggct tttctttcaa ttgcccgatc gcctttgata ttttcccaaa
     2101 ggataaaagc tagtccattc agaatcgagc cttaaagtac tcccatattg gctagcccca
     2161 gaattactcc agcgccgagg atgtggccaa agctagcggt gcccagcaca gcccctaaac
     2221 caaagccgcc aaagaagtta gaggaaggca tgggggtgcc cacattttgt tgtttgatgg
     2281 tcaatttacc aaaggcgatc gccaaaatgt tgcaagcaat catcacccca gcaactttag
     2341 ggctccagga cagggtggcg ggaacggcgg tggccaacaa aaagctatgc attgagattc
     2401 tccagaataa agacggtttt taaagggata gccccacgct aatgggggtc tttaaaaatc
     2461 tcatcttacg gggacgctct gcccctggga aaccaccgtt gcaatactta acaaattttc
     2521 gtttttagct tggcaaatgt ctttggcaaa attggttgat ctggcttaaa tcgtcagtta
     2581 tttgccctgg aatagtctgg ggacgggcaa ttctgatcag atttaccccc aacgcttccg
     2641 ccactttttg cttaaccaat tctcccccct gggcaccgga ggctttagtt accacccctt
     2701 gaatttgcca ttgttgccac agggcttttt ccaatggttc ggctacgggg gggcgcaaag
     2761 caatgatacg gtcggaagta aacccagcgg cgatcgcctg ggctagggct tggggatagg
     2821 gcagaatacg ggcaaataga acaaatctgg agccggtgag cgtgggtctc gcggtatcat
     2881 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     2941 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     3001 gcattggtaa ctgtcagacc aagtttacta atatatactt tagattgatt taaaacttca
     3061 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     3121 ttaacgtgag ttttcgttcc actgagcgtc agacccctta ataagatgat cttcttgaga
     3181 tcgttttggt ctgcgcgtaa tctcttgctc tgaaaacgaa aaaaccgcct tgcagggcgg
     3241 tttttcgaag gttctctgag ctaccaactc tttgaaccga ggtaactggc ttggaggagc
     3301 gcagtcacca aaacttgtcc tttcagttta gccttaaccg gcgcatgact tcaagactaa
     3361 ctcctctaaa tcaattacca gtggctgctg ccagtggtgc ttttgcatgt ctttccgggt
     3421 tggactcaag acgatagtta ccggataagg cgcagcggtc ggactgaacg gggggttcgt
     3481 gcatacagtc cagcttggag cgaactgcct acccggaact gagtgtcagg cgtggaatga
     3541 gacaaacgcg gccataacag cggaatgaca ccggtaaacc gaaaggcagg aacaggagag
     3601 cgcacgaggg agccgccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     3661 caccactgat ttgagcgtca gatttcgtga tgcttgtcag gggggcggag cctatggaaa
     3721 aacggctttg ccgcggccct ctcacttccc tgttaagtat cttcctggca tcttccagga
     3781 aatctccgcc ccgttcgtaa gccatttccg ctcgccgcag tcgaacgacc gagcgtagcg
     3841 agtcagtgag cgaggaagcg gaatatatcc tgtatcacat attctgctga cgcaccggtg
     3901 cagccttttt tctcctgcca catgaagcac ttcactgaca ccctcatcag tgccaacata
     3961 gtaagccagt atacactccg ctagcgctga ggtctgcctc gtgaagaagg tgttgctgac
     4021 tcataccagg cctgaatcgc cccatcatcc agccagaaag tgagggagcc acggttgatg
     4081 agagctttgt tgtaggtgga ccagttggtg attttgaact tttgctttgc cacggaacgg
     4141 tctgcgttgt cgggaagatg cggtgggtca atccctcggt ttggcgatcg ccttggtgtt
     4201 tgtcattgct ggtattgcct ttaaaatttc cgctgtaccc ttccaccaat ggacccccga
     4261 cgtttatgaa ggttctccca ccccggtggt ggcttttctt tcggtgggct ctaaggcggc
     4321 cggttttgcc gtagctatcc gattactagt gactgccttt ggtggtatta ccgatgagtg
     4381 gcacgttatt ttcaccgctc tggccgtgtt gagcatggtg ctgggcaacg tggtggcttt
     4441 agcccaaacc agcatgaaac ggatgttggc ctactcttcc atcggtcaag caggctttgt
     4501 gatgattggc ctagtggccg gcagtgaaga tggttacgcc agcatggttt tctacatgct
     4561 catctatctg tttatgaacc tgggggcgtt tagttgcatt attctcttca ccctccgcac
     4621 tggcagtgac caaattagtg attacgctgg tctgtaccac aaagacccct tgttaacctt
     4681 gggcttgagc atttgtttat tatccttggg gggcattcct cctctggcgg gctttttcgg
     4741 caaaatttac atcttctggg ccggttggca atcgggattg tatggcctag tcctacttgg
     4801 tctggttacc agtgtagttt ccatctacta ctacatccgg gtggtgaaaa tgatggtggt
     4861 gaaggagccc caggaaatgt ccgaagtaat caaaaattac ccggccatca aatggaattt
     4921 acccggcatg cgtcccctac aggtgggcat tgtcgctact ttggttgcta cctcgctggc
     4981 aggtattctg gctaatcccc tctttaacct cgccaccgat tccgtggtca gcaccaagat
     5041 gttgcagaca gccctccagc aaacaggaga aactccggcg atcgccattt cccatgattt
     5101 accctagggg tatcaggaaa tattgctttg caggcaaaag ccaatgagtg taactataga
     5161 aaccgattta aaggagactg cagacaattt tcgaaaaaac ccgcttcggc gggttttttt
     5221 atagctaaaa ccacaattca gcaaattgtg aacatcatca cgttcatctt tccctggttg
     5281 ccaatggccc attttcctgt cagtaacgag aaggtcgcgt attcaggcgc tttttagact
     5341 ggtcgtaatg aatcgggtaa gtttataata tacaaaggag gtagaaatgg tgagcaaggg
     5401 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     5461 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     5521 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccacctt
     5581 cggctacggc ctgcaatgct tcgcccgcta ccccgaccac atgaagctgc acgacttctt
     5641 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     5701 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     5761 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     5821 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
     5881 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     5941 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagctacca
     6001 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     6061 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaa
//
