LOCUS       sequence-183831-        7012 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TALEN Backbone for Phaeodactylum tricornutum_ NR promotor_NN.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7012)
  AUTHORS   Serif M, Lepetit B, Weissert K, Kroth PG, Rio Bartulos C
  TITLE     A fast and reliable strategy to generate TALEN-mediated gene 
            knockouts in the diatom Phaeodactylum tricornutum
  JOURNAL   Algal Research Volume 23, April 2017, Pages 186–195
REFERENCE   2  (bases 1 to 7012)
  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: "Algal 
            Research Volume 23, April 2017, Pages 186–195"
COMMENT     Sequence Label: pM9_NR_NN
FEATURES             Location/Qualifiers
     source          1..7012
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        501..519
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      521..530
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             530..595
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             602..622
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        join(1091..1096,1097..1114,1115..1121)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1175..1834
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1241..1260)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2176..2481
                     /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
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     2360..2379
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2785..3372
                     /codon_start=1
                     /product="nonspecific DNA cleavage domain of the FokI 
                     endonuclease (Li et al., 1992)"
                     /label=FokI cleavage domain
                     /note="must dimerize to be catalytically active"
                     /translation="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKV
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQR
                     YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     primer_bind     complement(3390..3407)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             3917..4486
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGFT
                     LREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNRR
                     LTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFTL
                     CGLDTALYDGTASDGEQALYMSMPCP"
     promoter        complement(4740..4758)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4845..4862)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5016..5604)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5096..5115)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5775..6566,6567..6635))
                     /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     6398..6417
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6636..6740)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6808..6826
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6864..6886)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6986..7005
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgcaccatat gcggaagtga ctgtaaacga gaagtgcacg aagccttttc ttgtgacgtc
       61 acaaaccgaa cagccctacg tggcgtgcga ctttccggca ctttggatat cgttgcccta
      121 tatgtattgg gtgtatacat gtcgtattcc aactacgaca gaaaacagtc agttacgggt
      181 attcgaactt ccggcacctg cagcgaggag ttttgtgtcc gtcgcacatc ctccgtgctt
      241 tcggcaccta cgtaggcggc aaacttcctg ccttccgccc gccttgcggc attccggctc
      301 cgggccagaa ttgcccgggt gttcacaatt ttgcctcctc acgaaaaaac gttctacttt
      361 gtattttggt cgggtttcgg atccttccag caactattct cattcaaagt caccacttgt
      421 gcgaacggaa ttcgatatca tcgactaatt cgagctctct ggctaactag agaacccact
      481 gcttactggc ttatcgaaat taatacgact cactataggg gccaccatgg actataagga
      541 ccacgacgga gactacaagg atcatgatat tgattacaaa gacgatgacg ataagatggc
      601 cccaaagaag aagcggaagg tcggtatcca cggagtccca gcagccgtag atttgagaac
      661 tttgggatat tcacagcagc agcaggaaaa gatcaagccc aaagtgaggt cgacagtcgc
      721 gcagcatcac gaagcgctgg tgggtcatgg gtttacacat gcccacatcg tagccttgtc
      781 gcagcaccct gcagcccttg gcacggtcgc cgtcaagtac caggacatga ttgcggcgtt
      841 gccggaagcc acacatgagg cgatcgtcgg tgtggggaaa cagtggagcg gagcccgagc
      901 gcttgaggcc ctgttgacgg tcgcgggaga gctgagaggg cctccccttc agctggacac
      961 gggccagttg ctgaagatcg cgaagcgggg aggagtcacg gcggtcgagg cggtgcacgc
     1021 gtggcgcaat gcgctcacgg gagcacccct caacctgaca gagaccgcgg ccgcattagg
     1081 caccccaggc tttacacttt atgcttccgg ctcgtataat gtgtggattt tgagttagga
     1141 tccgtcgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca ctggatatac
     1201 caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc agtcagttgc
     1261 tcaatgtacc tataaccaga ccgttcagct ggatattacg gcctttttaa agaccgtaaa
     1321 gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc tgatgaatgc
     1381 tcatccggaa ttccgtatgg caatgaaaga cggtgagctg gtgatatggg atagtgttca
     1441 cccttgttac accgttttcc atgagcaaac tgaaacgttt tcatcgctct ggagtgaata
     1501 ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt gttacggtga
     1561 aaacctggcc tatttcccta aagggtttat tgagaatatg tttttcgtct cagccaatcc
     1621 ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat atggacaact tcttcgcccc
     1681 cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc cgctggcgat
     1741 tcaggttcat catgccgttt gtgatggctt ccatgtcggc agaatgctta atgaattaca
     1801 acagtactgc gatgagtggc agggcggggc gtaaagatct ggatccggct tactaaaagc
     1861 cagataacag tatgcgtatt tgcgcgctga tttttgcggt ataagaatat atactgatat
     1921 gtatacccga agtatgtcaa aaagaggtat gctatgaagc agcgtattac agtgacagtt
     1981 gacagcgaca gctatcagtt gctcaaggca tatatgatgt caatatctcc ggtctggtaa
     2041 gcacaaccat gcagaatgaa gcccgtcgtc tgcgtgccga acgctggaaa gcggaaaatc
     2101 aggaagggat ggctgaggtc gcccggttta ttgaaatgaa cggctctttt gctgacgaga
     2161 acaggggctg gtgaaatgca gtttaaggtt tacacctata aaagagagag ccgttatcgt
     2221 ctgtttgtgg atgtacagag tgatattatt gacacgcccg ggcgacggat ggtgatcccc
     2281 ctggccagtg cacgtctgct gtcagataaa gtctcccgtg aactttaccc ggtggtgcat
     2341 atcggggatg aaagctggcg catgatgacc accgatatgg ccagtgtgcc ggtctccgtt
     2401 atcggggaag aagtggctga tctcagccac cgcgaaaatg acatcaaaaa cgccattaac
     2461 ctgatgttct ggggaatata aatgtcaggc tcccttatac acagccagtc tgcaggtcga
     2521 cggtctcgac tcacgcctga gcaggtagtg gctattgcat ccaacaacgg gggcagaccc
     2581 gcactggagt caatcgtggc ccagctttcg aggccggacc ccgcgctggc cgcactcact
     2641 aatgatcatc ttgtagcgct ggcctgcctc ggcggacgac ccgccttgga tgcggtgaag
     2701 aaggggctcc cgcacgcgcc tgcattgatt aagcggacca acagaaggat tcccgagagg
     2761 acatcacatc gagtggcagg ttcccaactc gtgaagagtg aacttgagga gaaaaagtcg
     2821 gagctgcggc acaaattgaa atacgtaccg catgaataca tcgaacttat cgaaattgct
     2881 aggaactcga ctcaagacag aatccttgag atgaaggtaa tggagttctt tatgaaggtt
     2941 tatggatacc gagggaagca tctcggtgga tcacgaaaac ccgacggagc aatctatacg
     3001 gtggggagcc cgattgatta cggagtgatc gtcgacacga aagcctacag cggtgggtac
     3061 aatcttccca tcgggcaggc agatgagatg caacgttatg tcgaagaaaa tcagaccagg
     3121 aacaaacaca tcaatccaaa tgagtggtgg aaagtgtatc cttcatcagt gaccgagttt
     3181 aagtttttgt ttgtctctgg gcatttcaaa ggcaactata aggcccagct cacacggttg
     3241 aatcacatta cgaactgcaa tggtgcggtt ttgtccgtag aggaactgct cattggtgga
     3301 gaaatgatca aagcgggaac tctgacactg gaagaagtca gacgcaagtt taacaatggc
     3361 gagatcaatt tccgctcata aaaaatcagc ctcgactgtg ccttctagtt gccagccatc
     3421 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa gcttcaattt actttaagac
     3481 tagcgcaatt tttttcttac attagatgaa cataaaacga cgatgaggat cactgttagg
     3541 gaaacgatga actacgctga ggacttccta ttattcgtcc tttgatggcg tggttgttct
     3601 ttggtttgcg gccgagcgca acgaccaagc cctgtaccaa aaacgaagtt gataatcaga
     3661 tatttgagtt gtaacaagaa actaaagcat tattttagca gtagcacgaa tcaggatcta
     3721 aaatgaacgc atccggagag attcaaaatt caatctgttt ggacctggat aagacacaag
     3781 agcgacatcc tgacatgaac gccgtaaaca gcaaatcctg gttgaacacg tatccttttg
     3841 ggggcctccc gctacgacgc tcgctccagc tggggcttcc ttactataca cagcgcgcat
     3901 atttcacggt tgccagatga ccactcttga cgacacggct taccggtacc gcaccagtgt
     3961 cccgggggac gccgaggcca tcgaggcact ggatgggtcc ttcaccaccg acaccgtctt
     4021 ccgcgtcacc gccaccgggg acggcttcac cctgcgggag gtgccggtgg acccgcccct
     4081 gaccaaggtg ttccccgacg acgaatcgga cgacgaatcg gacgacgggg aggacggcga
     4141 cccggactcc cggacgttcg tcgcgtacgg ggacgacggc gacctggcgg gcttcgtggt
     4201 cgtctcgtac tccggctgga accgccggct gaccgtcgag gacatcgagg tcgccccgga
     4261 gcaccggggg cacggggtcg ggcgcgcgtt gatggggctc gcgacggagt tcgcccgcga
     4321 gcggggcgcc gggcacctct ggctggaggt caccaacgtc aacgcaccgg cgatccacgc
     4381 gtaccggcgg atggggttca ccctctgcgg cctggacacc gccctgtacg acggcaccgc
     4441 ctcggacggc gagcaggcgc tctacatgag catgccctgc ccctgaaaac cttccttaaa
     4501 aatttaattt tcattagttg cagtcactcc gctttggttt cacagtcagg aataacacta
     4561 gctcgtcttc accatggatg ccaatctcgc ctattcatgg tgtataaaag ttcaacatcc
     4621 aaagctagaa cttttggaaa gagaaagaat atccgaatag ggcacggcgt gccgtattgt
     4681 tggagtggac tagcagaaag tgaggaaggc acaggatgag ttttctcgag gccggactcc
     4741 ctatagtgag tcgtattaat ttcgataagc caggttaacc tgcattaatg aatcggccaa
     4801 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     4861 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     4921 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     4981 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5041 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5101 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5161 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5221 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5281 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5341 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5401 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     5461 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     5521 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5581 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5641 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5701 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     5761 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     5821 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     5881 ttaccatctg gccccagtgc tgcaatgata ccgcgggacc cacgctcacc ggctccagat
     5941 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     6001 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     6061 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     6121 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     6181 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     6241 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     6301 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     6361 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     6421 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6481 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6541 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6601 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     6661 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6721 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     6781 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg
     6841 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
     6901 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     6961 gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag ag
//
