LOCUS       sequence-183795-        7058 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TALEN Backbone for Phaeodactylum tricornutum_ fcpA promotor_NI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7058)
  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 7058)
  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_fcpA_NI
FEATURES             Location/Qualifiers
     source          1..7058
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        517..535
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      537..546
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             546..611
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             618..638
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        join(1107..1112,1113..1130,1131..1137)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1191..1850
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1257..1276)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2192..2497
                     /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     2376..2395
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2801..3388
                     /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(3406..3423)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             4057..4431
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     promoter        complement(4683..4701)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4788..4805)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4959..5547)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5039..5058)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5718..6509,6510..6578))
                     /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     6341..6360
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6579..6683)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6751..6769
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6807..6829)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6929..6948
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        7029..7047
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     7029..7046
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 cgggctgcag gacgcaatgg aggattatca ccgcaaaaat gaacttcgaa aaaaactttc
       61 gagcgaccat ggaaaaggag gatcagattc agattacaac agtggattgc tctggtagca
      121 aatatcttct gctagattgg ctcatggtcg gttttggacg ttcgaagctc accgtcaaaa
      181 gaaacaaaag agaagaatga cgtcttcgtg acgtagaatc tacgactgta ctcggatctg
      241 ggaaatgaat tgactcacgg tcttcttcga gtcctgttac aggcccttgg tccgaacccc
      301 cacacgattt ttgcaccaaa gatttgcttc aatttgctgg atgttttgac tgcaagatca
      361 gctggcctag caagagtgct cgtgttgctt cgtcgggaat ccctacgaat ttcagttctg
      421 cacaaatttg tctgccgttt cgagaattcg atatcatcga ctaattcgag ctctctggct
      481 aactagagaa cccactgctt actggcttat cgaaattaat acgactcact ataggggcca
      541 ccatggacta taaggaccac gacggagact acaaggatca tgatattgat tacaaagacg
      601 atgacgataa gatggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag
      661 ccgtagattt gagaactttg ggatattcac agcagcagca ggaaaagatc aagcccaaag
      721 tgaggtcgac agtcgcgcag catcacgaag cgctggtggg tcatgggttt acacatgccc
      781 acatcgtagc cttgtcgcag caccctgcag cccttggcac ggtcgccgtc aagtaccagg
      841 acatgattgc ggcgttgccg gaagccacac atgaggcgat cgtcggtgtg gggaaacagt
      901 ggagcggagc ccgagcgctt gaggccctgt tgacggtcgc gggagagctg agagggcctc
      961 cccttcagct ggacacgggc cagttgctga agatcgcgaa gcggggagga gtcacggcgg
     1021 tcgaggcggt gcacgcgtgg cgcaatgcgc tcacgggagc acccctcaac ctgacagaga
     1081 ccgcggccgc attaggcacc ccaggcttta cactttatgc ttccggctcg tataatgtgt
     1141 ggattttgag ttaggatccg tcgagatttt caggagctaa ggaagctaaa atggagaaaa
     1201 aaatcactgg atataccacc gttgatatat cccaatggca tcgtaaagaa cattttgagg
     1261 catttcagtc agttgctcaa tgtacctata accagaccgt tcagctggat attacggcct
     1321 ttttaaagac cgtaaagaaa aataagcaca agttttatcc ggcctttatt cacattcttg
     1381 cccgcctgat gaatgctcat ccggaattcc gtatggcaat gaaagacggt gagctggtga
     1441 tatgggatag tgttcaccct tgttacaccg ttttccatga gcaaactgaa acgttttcat
     1501 cgctctggag tgaataccac gacgatttcc ggcagtttct acacatatat tcgcaagatg
     1561 tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg gtttattgag aatatgtttt
     1621 tcgtctcagc caatccctgg gtgagtttca ccagttttga tttaaacgtg gccaatatgg
     1681 acaacttctt cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc
     1741 tgatgccgct ggcgattcag gttcatcatg ccgtttgtga tggcttccat gtcggcagaa
     1801 tgcttaatga attacaacag tactgcgatg agtggcaggg cggggcgtaa agatctggat
     1861 ccggcttact aaaagccaga taacagtatg cgtatttgcg cgctgatttt tgcggtataa
     1921 gaatatatac tgatatgtat acccgaagta tgtcaaaaag aggtatgcta tgaagcagcg
     1981 tattacagtg acagttgaca gcgacagcta tcagttgctc aaggcatata tgatgtcaat
     2041 atctccggtc tggtaagcac aaccatgcag aatgaagccc gtcgtctgcg tgccgaacgc
     2101 tggaaagcgg aaaatcagga agggatggct gaggtcgccc ggtttattga aatgaacggc
     2161 tcttttgctg acgagaacag gggctggtga aatgcagttt aaggtttaca cctataaaag
     2221 agagagccgt tatcgtctgt ttgtggatgt acagagtgat attattgaca cgcccgggcg
     2281 acggatggtg atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact
     2341 ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag
     2401 tgtgccggtc tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat
     2461 caaaaacgcc attaacctga tgttctgggg aatataaatg tcaggctccc ttatacacag
     2521 ccagtctgca ggtcgacggt ctcgactcac gcctgagcag gtagtggcta ttgcatccaa
     2581 catcgggggc agacccgcac tggagtcaat cgtggcccag ctttcgaggc cggaccccgc
     2641 gctggccgca ctcactaatg atcatcttgt agcgctggcc tgcctcggcg gacgacccgc
     2701 cttggatgcg gtgaagaagg ggctcccgca cgcgcctgca ttgattaagc ggaccaacag
     2761 aaggattccc gagaggacat cacatcgagt ggcaggttcc caactcgtga agagtgaact
     2821 tgaggagaaa aagtcggagc tgcggcacaa attgaaatac gtaccgcatg aatacatcga
     2881 acttatcgaa attgctagga actcgactca agacagaatc cttgagatga aggtaatgga
     2941 gttctttatg aaggtttatg gataccgagg gaagcatctc ggtggatcac gaaaacccga
     3001 cggagcaatc tatacggtgg ggagcccgat tgattacgga gtgatcgtcg acacgaaagc
     3061 ctacagcggt gggtacaatc ttcccatcgg gcaggcagat gagatgcaac gttatgtcga
     3121 agaaaatcag accaggaaca aacacatcaa tccaaatgag tggtggaaag tgtatccttc
     3181 atcagtgacc gagtttaagt ttttgtttgt ctctgggcat ttcaaaggca actataaggc
     3241 ccagctcaca cggttgaatc acattacgaa ctgcaatggt gcggttttgt ccgtagagga
     3301 actgctcatt ggtggagaaa tgatcaaagc gggaactctg acactggaag aagtcagacg
     3361 caagtttaac aatggcgaga tcaatttccg ctcataaaaa atcagcctcg actgtgcctt
     3421 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaagctt
     3481 cagaagcgtg ctatcgaact caaccaggga cgtgcggcac aaatgggcat ccttgctctc
     3541 atggtgcacg aacagttggg agtctctatc cttccttaaa aatttaattt tcattagttg
     3601 cagtcactcc gctttggttt cacagtcagg aataacacta gctcgtcttc accatggatg
     3661 ccaatctcgc ctattcatgg tgtataaaag ttcaacatcc aaagctagaa cttttggaaa
     3721 gagaaagaat atccgaatag ggcacggcgt gccgtattgt tggagtggac tagcagaaag
     3781 tgaggaaggc acaggatgag ttttctcgag acataccttc agcgtcgtct tcactgtcac
     3841 agtcaactga cagtaatcgt tgatccggag agattcaaaa ttcaatctgt ttggacctgg
     3901 ataagacaca agagcgacat cctgacatga acgccgtaaa cagcaaatcc tggttgaaca
     3961 cgtatccttt tgggggcctc ccgctacgac gctcgctcca gctggggctt ccttactata
     4021 cacagcgcgc atatttcacg gttgccagat gtcaagatgg ccaagttgac cagtgccgtt
     4081 ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg
     4141 ttctcccggg acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg
     4201 ttcatcagcg cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg
     4261 cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac
     4321 gcctccgggc cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg
     4381 cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg agcaggactg aaccttcctt
     4441 aaaaatttaa ttttcattag ttgcagtcac tccgctttgg tttcacagtc aggaataaca
     4501 ctagctcgtc ttcaccatgg atgccaatct cgcctattca tggtgtataa aagttcaaca
     4561 tccaaagcta gaacttttgg aaagagaaag aatatccgaa tagggcacgg cgtgccgtat
     4621 tgttggagtg gactagcaga aagtgaggaa ggcacaggat gagttttctc gaggccggac
     4681 tccctatagt gagtcgtatt aatttcgata agccaggtta acctgcatta atgaatcggc
     4741 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4801 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4861 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4921 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4981 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     5041 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     5101 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     5161 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5221 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5281 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5341 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     5401 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5461 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5521 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     5581 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5641 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     5701 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     5761 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     5821 ggcttaccat ctggccccag tgctgcaatg ataccgcggg acccacgctc accggctcca
     5881 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5941 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     6001 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     6061 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     6121 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     6181 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     6241 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     6301 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     6361 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6421 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6481 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6541 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     6601 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6661 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     6721 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc
     6781 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca
     6841 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt
     6901 ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac
     6961 catatggaca tattgtcgtt agaacgcggc tacaattaat acataacctt atgtatcata
     7021 cacatacgat ttaggtgaca ctatagaacc agatcccc
//
