LOCUS       sequence-423179-        8396 bp    DNA     circular SYN 15-APR-2025
DEFINITION  For creating CyanoTag vectors by Golden Gate cloning (BsaI version).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8396)
  TITLE     CyanoTag plasmids, for native C-terminal fluorescent tagging of 
            proteins in cyanobacteria (S. elongatus 7942)
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8396)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pLM434
FEATURES             Location/Qualifiers
     source          1..8396
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             718..783
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     terminator      791..861
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      877..904
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(1043..1062)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1054..1869
                     /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             1054..1869
                     /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(1127..1146)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(2882..4165)
                     /codon_start=1
                     /gene="codA"
                     /product="E. coli cytosine deaminase"
                     /label=codA
                     /note="confers sensitivity to 5-fluorocytosine"
                     /translation="MSNNALQTIINARLPGEEGLWQIHLQDGKISAIDAQSGVMPITEN
                     SLDAEQGLVIPPFVEPHIHLDTTQTAGQPNWNQSGTLFEGIERWAERKALLTHDDVKQR
                     AWQTLKWQIANGIQHVRTHVDVSDATLTALKAMLEVKQEVAPWIDLQIVAFPQEGILSY
                     PNGEALLEEALRLGADVVGAIPHFEFTREYGVESLHKTFALAQKYDRLIDVHCDEIDDE
                     QSRFVETVAALAHHEGMGARVTASHTTAMHSYNGAYTSRLFRLLKMSGINFVANPLVNI
                     HLQGRFDTYPKRRGITRVKEMLESGINVCFGHDDVFDPWYPLGTANMLQVLHMGLHVCQ
                     LMGYGQINDGLNLITHHSARTLNLQDYGIAAGNSANLIILPAENGFDALRRQVPVRYSV
                     RGGKVIASTQPAQTTVYLEQPEAIDYKR"
     CDS             5180..5245
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5578..5883
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     rep_origin      complement(5947..6535)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6027..6046)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6706..7497,7498..7566))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7329..7348
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7567..7671)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             8011..8316
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
ORIGIN
        1 atggttagta agggtgagga agacaatatg gcaagcctgc cggcaacgca cgaactgcat
       61 atctttggtt cgatcaacgg cgtggacttt gacatggtcg gccaggggac tggtaacccg
      121 aacgatggct acgaggaact gaacctgaaa tcgacaaaag gcgatctgca attctcgcca
      181 tggattctcg tgccgcatat tgggtacggc tttcaccagt atttgccgta tccagatggc
      241 atgagtccgt ttcaagccgc tatggtcgat ggtagcggct atcaggtgca tcgcactatg
      301 cagtttgaag acggcgccag tttgacagtc aattaccgtt acacttatga aggctcgcat
      361 attaaaggtg aggcgcaagt taagggtacc gggtttcccg ccgacggtcc cgtgatgact
      421 aatagcctga ccgcggctga ttggtgccgt agcaaaaaaa cctacccgaa cgacaaaacc
      481 atcatttcca cgtttaaatg gagctatact acaggtaacg ggaagcgcta tcgctcgacg
      541 gcgcgcacta catacacgtt cgcgaaaccg atggccgcga attacctcaa aaaccagccg
      601 atgtatgtgt ttcgtaaaac cgagctgaaa catagcaaga cagaactgaa ctttaaagag
      661 tggcagaaag cattcacaga cgtcatgggc atggatgaac tgtacaaggg cggtggcgat
      721 tacaaagatc acgatggcga ttacaaagat cacgacatcg actataagga tgacgatgat
      781 aagtaggctt caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg
      841 tttttgtcgg tgaacgctct ctactagagt cacactggct caccttcggg tgggcctttc
      901 tgcgggaatt cgattgatcc gtcgacctgc aggggggggg gggaaagcca cgttgtgtct
      961 caaaatctct gatgttacat tgcacaagat aaaaatatat catcatgaac aataaaactg
     1021 tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtct
     1081 tgctcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct
     1141 cgcgataatg tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg
     1201 ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg
     1261 gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt
     1321 actcctgatg atgcatggtt actcaccact gcgatccccg ggaaaacagc attccaggta
     1381 ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc
     1441 cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc
     1501 gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag
     1561 cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataagctttt gccattctca
     1621 ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg
     1681 aaattaatag gttgtattga tgttggacgc gtcggaatcg cagaccgata ccaggatctt
     1741 gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa
     1801 aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag
     1861 tttttctaat cagaattggt taattggttg taacactggc agagcattac gctgacttga
     1921 cgggacggcg gctttgttga ataaatcgaa cttttgctga gttgaaggat cagatcacgc
     1981 atcttcccga caacgcagac cgttccgtgg caaagcaaaa gttcaaaatc accaactggt
     2041 ccacctacaa caaagctctc atcaaccgtg gctccctcac tttctggctg gatgatgggg
     2101 cgattcaggc ctggtatgag ccagcaacac cttcttcacg aggcagacct cagcgctcct
     2161 ccaccgctgc agttcactta caccgcttct caacccggta cgcaccagaa aatcattgat
     2221 atggccatga atggcgttgg atgccgggca acagcccgca ttatgggcgt tggcctcaac
     2281 acgattttac gtcacttaaa aaactcaggc cgcagtcggt aacctcgcgc atacagccgg
     2341 gcagtgacgt catcgtctgc gcggaaatgg acgaacagtg gggctatgtc ggggctaaat
     2401 cgcgccagcg ctggctgttt tacgcgtatg acagtctccg gaagacggtt gttgcgcacg
     2461 tattcggtga acgcactatg gcgacgctgg ggcgtcttat gagcctgctg tcaccctttg
     2521 acgtggtgat atggatgacg gatggctggc cgctgtatga atcccgcctg aagggaaagc
     2581 tgcacgtaat cagcaagcga tatacgcagc gaattgagcg gcataacctg aatctgaggc
     2641 agcacctggc acggctggga cggaagtcgc tgtcgttctc aaaatcggtg gagctgcatg
     2701 acaaagtcat cgggcattat ctgaacataa aacactatca ataagttgga atcattacca
     2761 aaaggttagg aatacggtta gccatttgcc tgcttttata tagttcatat gggattcacc
     2821 tttatgttga taagaaataa aagaaaatgc caataggata tcggcatttt cttttgcgtt
     2881 ttcaacgttt gtaatcgatg gcttctggct gctccagata tacggtggtt tgtgccggtt
     2941 gtgtgctggc aatcaccttg ccgccacgta ccgaataacg taccggaacc tgacggcgca
     3001 gcgcatcaaa cccattttca gccggcagga taatcaggtt ggcgctgttt ccggcggcaa
     3061 tgccgtaatc ctgcaaattc aacgtccttg cgctgtggtg ggtgattaaa ttcaggccat
     3121 cgttaatctg cccgtagccc atcaactggc aaacatgcag ccccatatgc agcacttgca
     3181 gcatattcgc cgttcccagc ggataccacg gatcgaagac atcatcgtga ccaaagcaga
     3241 cgttaatgcc cgattccagc atctctttaa cgcgcgtgat gccgcgacgt tttggatacg
     3301 tatcgaaacg tccttgcaga tgaatattga ccagcgggtt ggcgacaaag ttaataccgg
     3361 acattttcag caagcggaac aggcgtgagg tatacgcccc gttataggag tgcattgccg
     3421 tggtgtggct ggcggtgacg cgcgcgccca tgccttcatg gtgcgccagg gcagcaacgg
     3481 tttcgacaaa gcgcgactgc tcgtcatcga tctcatcaca gtgaacgtcg atgagacggt
     3541 cgtatttttg cgccagggcg aaggttttat gcagcgattc cacgccgtat tcacgggtaa
     3601 attcaaaatg cggaatcgcc cccactacat ctgcccctaa gcgtaacgcc tcttccagca
     3661 acgcttcacc gttgggatac gacaaaatcc cttcctgagg gaaggcgacg atttgcagat
     3721 caatccacgg cgcgacttcc tgcttcactt ccagcattgc tttcagcgca gttagcgttg
     3781 catccgaaac atcgacatgg gtacgcacat gctgaatgcc gttggcaatc tgccatttca
     3841 gcgtttgcca tgcgcgttgt ttcacatcgt catgggttaa taacgctttg cgctcggccc
     3901 agcgttcaat gccttcaaac agcgtgccgg actgattcca gttcggttgt ccggcggttt
     3961 gcgtggtgtc caggtgaata tgtggctcca caaacggcgg tataactaaa ccttgttcgg
     4021 catccaggct gttttcagtt atgggcatca cgccggattg cgcatcaatg gcgctgattt
     4081 ttccgtcctg cagatgaatc tgccacagcc cctcttcgcc tggtaaccgg gcgttaataa
     4141 ttgtttgtaa agcgttattc gacatcgttc atgtctcctt ttttatgtac tgtgttagcg
     4201 gtctgcttct tccagccctc ctgtttgaag atggcaagtt agttacgcac aataaaaaaa
     4261 gacctaaaat atgtaagggg tgacgccaaa gtatacactt tgccctttac acattttagg
     4321 tcttgcctgc tttatcagta acaaacccgc gcgatttact tttcgacctc attctattag
     4381 attctcgttt ggattgcaac tggtctattt tcctcttttg tttgatagaa aatcataaaa
     4441 ggatttgcag actacgggcc taaaggttag taagggtgag gaagacaata tggcaagcct
     4501 gccggcaacg cacgaactgc atatctttgg ttcgatcaac ggcgtggact ttgacatggt
     4561 cggccagggg actggtaacc cgaacgatgg ctacgaggaa ctgaacctga aatcgacaaa
     4621 aggcgatctg caattctcgc catggattct cgtgccgcat attgggtacg gctttcacca
     4681 gtatttgccg tatccagatg gcatgagtcc gtttcaagcc gctatggtcg atggtagcgg
     4741 ctatcaggtg catcgcacta tgcagtttga agacggcgcc agtttgacag tcaattaccg
     4801 ttacacttat gaaggctcgc atattaaagg tgaggcgcaa gttaagggta ccgggtttcc
     4861 cgccgacggt cccgtgatga ctaatagcct gaccgcggct gattggtgcc gtagcaaaaa
     4921 aacctacccg aacgacaaaa ccatcatttc cacgtttaaa tggagctata ctacaggtaa
     4981 cgggaagcgc tatcgctcga cggcgcgcac tacatacacg ttcgcgaaac cgatggccgc
     5041 gaattacctc aaaaaccagc cgatgtatgt gtttcgtaaa accgagctga aacatagcaa
     5101 gacagaactg aactttaaag agtggcagaa agcattcaca gacgtcatgg gcatggatga
     5161 actgtacaag ggcggtggcg attacaaaga tcacgatggc gattacaaag atcacgacat
     5221 cgactataag gatgacgatg ataagtagtg agacctaaaa gccagataac agtatgcgta
     5281 tttgcgcgct gatttttgcg gtataagaat atatactgat atgtataccc gaagtatgtc
     5341 aaaaagaggt atgctatgaa gcagcgtatt acagtgacag ttgacagcga cagctatcag
     5401 ttgctcaagg catatatgat gtcaatatct ccggtctggt aagcacaacc atgcagaatg
     5461 aagcccgtcg tctgcgtgcc gaacgctgga aagcggaaaa tcaggaaggg atggctgagg
     5521 tcgcccggtt tattgaaatg aacggctctt ttgctgacga gaacaggggc tggtgaaatg
     5581 cagtttaagg tttacaccta taaaagagag agccgttatc gtctgtttgt ggatgtacag
     5641 agtgatatta ttgacacgcc cgggcgacgg atggtgatcc ccctggccag tgcacgtctg
     5701 ctgtcagata aagtcccccg tgaactttac ccggtggtgc atatcgggga tgaaagctgg
     5761 cgcatgatga ccaccgatat ggccagtgtg ccggtttccg ttatcgggga agaagtggct
     5821 gatctcagcc accgcgaaaa tgacatcaaa aacgccatta acctgatgtt ctggggaata
     5881 taaatgtcag gctcccttat acacagccag tctgcaggtc gaccatagtg gtctcacttg
     5941 agactctttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     6001 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     6061 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     6121 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     6181 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     6241 cggtaactat cgtcttgagc ccaacccggt aagacacgac ttatcgccac tggcagcagc
     6301 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     6361 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     6421 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     6481 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     6541 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     6601 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     6661 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     6721 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctggctccc
     6781 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     6841 accgcgtgac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     6901 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     6961 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     7021 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     7081 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     7141 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     7201 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     7261 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     7321 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     7381 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     7441 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     7501 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     7561 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     7621 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc ggagtcagac
     7681 ttgagaccta aaagccagat aacagtatgc gtatttgcgc gctgattttt gcggtataag
     7741 aatatatact gatatgtata cccgaagtat gtcaaaaaga ggtatgctat gaagcagcgt
     7801 attacagtga cagttgacag cgacagctat cagttgctca aggcatatat gatgtcaata
     7861 tctccggtct ggtaagcaca accatgcaga atgaagcccg tcgtctgcgt gccgaacgct
     7921 ggaaagcgga aaatcaggaa gggatggctg aggtcgcccg gtttattgaa atgaacggct
     7981 cttttgctga cgagaacagg ggctggtgaa atgcagttta aggtttacac ctataaaaga
     8041 gagagccgtt atcgtctgtt tgtggatgta cagagtgata ttattgacac gcccgggcga
     8101 cggatggtga tccccctggc cagtgcacgt ctgctgtcag ataaagtccc ccgtgaactt
     8161 tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga tgaccaccga tatggccagt
     8221 gtgccggttt ccgttatcgg ggaagaagtg gctgatctca gccaccgcga aaatgacatc
     8281 aaaaacgcca ttaacctgat gttctgggga atataaatgt caggctccct tatacacagc
     8341 cagtctgcag gtcgaccata gtggtctcag gcgatctggg tggcagcggc ggccgc
//
