LOCUS       sequence-174536-        6856 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TALEN-mediated mutagenesis of Exon 2 in NvFP-7R.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6856)
  AUTHORS   Ikmi A, McKinney SA, Delventhal KM, Gibson MC
  TITLE     TALEN and CRISPR/Cas9-mediated genome editing in the early-branching
            metazoan Nematostella vectensis.
  JOURNAL   Nat Commun. 2014 Nov 24;5:5486. doi: 10.1038/ncomms6486.
  PUBMED    25417547
REFERENCE   2  (bases 1 to 6856)
  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: "Nat 
            Commun."; date: "2014-11-24"; volume: "5:5486. doi"; pages: " 
            10.1038/ncomms6486"
COMMENT     Sequence Label: pCS2TAL3_DD_NvFP-7R_Ex2-LEFT
FEATURES             Location/Qualifiers
     source          1..6856
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      85..94
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             97..117
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             118..183
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2281..2868
                     /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
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQD
                     YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     polyA_signal    2901..3035
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2925..2944)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2979..2998
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3152..3172)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3152..3170)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3191..3207)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3191..3207)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3204..3226)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3215..3231
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3239..3245,3246..3263,3264..3269))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3284..3305
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3422..3439)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3593..4181)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3673..3692)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4352..5143,5144..5212))
                     /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     4975..4994
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5213..5317)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5343..5798)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5480..5501)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5692..5711
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5872..6856
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atctgccacc atggctccaa agaagaagcg taaggtagac
      121 tacaaagacc atgacggtga ttataaagat catgacatcg attacaagga tgacgatgac
      181 aagggtaccg tggatctacg cacgctcggc tacagtcagc agcagcaaga gaagatcaaa
      241 ccgaaggtgc gttcgacagt ggcgcagcac cacgaggcac tggtgggcca tgggtttaca
      301 cacgcgcaca tcgttgcgct cagccaacac ccggcagcgt tagggaccgt cgctgtcacg
      361 tatcagcaca taatcacggc gttgccagag gcgacacacg aagacatcgt tggcgtcggc
      421 aaacagtggt ccggcgcacg cgccctggag gccttgctca cggatgcggg ggagttgaga
      481 ggtccgccgt tacagttgga cacaggccaa cttgtgaaga ttgcaaaacg tggcggcgtg
      541 accgcaatgg aggcagtgca tgcatcgcgc aatgcactga cgggtgcccc cctgaacctg
      601 accccggacc aagtggtggc tatcgccagc aacattggcg gcaagcaagc gctcgaaacg
      661 gtgcagcggc tgttgccggt gctgtgccag gaccatggcc tgaccccgga ccaagtggtg
      721 gctatcgcca gcaacaatgg cggcaagcaa gcgctcgaaa cggtgcagcg gctgttgccg
      781 gtgctgtgcc aggaccatgg cctgaccccg gaccaagtgg tggctatcgc cagcaacggt
      841 ggcggcaagc aagcgctcga aacggtgcag cggctgttgc cggtgctgtg ccaggaccat
      901 ggcctgaccc cggaccaagt ggtggctatc gccagcaaca ttggcggcaa gcaagcgctc
      961 gaaacggtgc agcggctgtt gccggtgctg tgccaggacc atggcctgac cccggaccaa
     1021 gtggtggcta tcgccagcaa cattggcggc aagcaagcgc tcgaaacggt gcagcggctg
     1081 ttgccggtgc tgtgccagga ccatggcctg accccggacc aagtggtggc tatcgccagc
     1141 aacattggcg gcaagcaagc gctcgaaacg gtgcagcggc tgttgccggt gctgtgccag
     1201 gaccatggcc tgaccccgga ccaagtggtg gctatcgcca gcaacaatgg cggcaagcaa
     1261 gcgctcgaaa cggtgcagcg gctgttgccg gtgctgtgcc aggaccatgg cctgaccccg
     1321 gaccaagtgg tggctatcgc cagcaacatt ggcggcaagc aagcgctcga aacggtgcag
     1381 cggctgttgc cggtgctgtg ccaggaccat ggcctgaccc cggaccaagt ggtggctatc
     1441 gccagcaacg gtggcggcaa gcaagcgctc gaaacggtgc agcggctgtt gccggtgctg
     1501 tgccaggacc atggcctgac cccggaccaa gtggtggcta tcgccagcaa caatggcggc
     1561 aagcaagcgc tcgaaacggt gcagcggctg ttgccggtgc tgtgccagga ccatggcctg
     1621 accccggacc aagtggtggc tatcgccagc cacgatggcg gcaagcaagc gctcgaaacg
     1681 gtgcagcggc tgttgccggt gctgtgccag gaccatggcc tgaccccgga ccaagtggtg
     1741 gctatcgcca gcaacggtgg cggcaagcaa gcgctcgaaa cggtgcagcg gctgttgccg
     1801 gtgctgtgcc aggaccatgg cctgaccccg gaccaagtgg tggctatcgc cagcaacatt
     1861 ggcggcaagc aagcgctcga aacggtgcag cggctgttgc cggtgctgtg ccaggaccat
     1921 ggcctgaccc cggaccaagt ggtggctatc gccagcaacg gtggcggcaa gcaagcgctc
     1981 gaaacggtgc agcggctgtt gccggtgctg tgccaggacc atggcctgac cccggaccaa
     2041 gtggtggcta tcgccagcaa caatggcggc aagcaagcgc tcgaaagcat tgtggcccag
     2101 ctgagccggc ctgatccggc gttggccgcg ttgaccaacg accacctcgt cgccttggcc
     2161 tgcctcggcg gacgtcctgc catggatgca gtgaaaaagg gattgccgca cgcgccggaa
     2221 ttgatcagaa gagtcaatcg ccgtattggc gaacgcacgt cccatcgcgt tgccggatcc
     2281 caactagtca aaagtgaact ggaggagaag aaatctgaac ttcgtcataa attgaaatat
     2341 gtgcctcatg aatatattga attaattgaa attgccagaa attccactca ggatagaatt
     2401 cttgaaatga aggtaatgga attttttatg aaagtttatg gatatagagg taaacatttg
     2461 ggtggatcaa ggaaaccgga cggagcaatt tatactgtcg gatctcctat tgattacggt
     2521 gtgatcgtgg atactaaagc ttatagcgga ggttataatc tgccaattgg ccaagcagat
     2581 gaaatgcaag actatgtcga agaaaatcaa acacgaaaca aacatatcaa ccctaatgaa
     2641 tggtggaaag tctatccatc ttctgtaacg gaatttaagt ttttatttgt gagtggtcac
     2701 tttaaaggaa actacaaagc tcagcttaca cgattaaatc atatcactaa ttgtaatgga
     2761 gctgttctta gtgtagaaga gcttttaatt ggtggagaaa tgattaaagc cggcacatta
     2821 accttagagg aagtgcgccg gaaatttaat aacggcgaga taaactttta atctagaact
     2881 atagtgagtc gtattacgta gatccagaca tgataagata cattgatgag tttggacaaa
     2941 ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt
     3001 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta
     3061 tgtttcaggt tcagggggag gtgtgggagg ttttttaatt cgcggccgcg gcgccaatgc
     3121 attgggcccg gtacgtaccc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg
     3181 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     3241 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     3301 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     3361 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     3421 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     3481 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     3541 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     3601 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     3661 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     3721 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     3781 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     3841 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     3901 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     3961 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     4021 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     4081 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     4141 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     4201 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     4261 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     4321 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     4381 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     4441 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     4501 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     4561 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     4621 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     4681 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     4741 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     4801 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     4861 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     4921 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     4981 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     5041 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     5101 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     5161 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     5221 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     5281 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     5341 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     5401 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     5461 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     5521 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     5581 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     5641 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     5701 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     5761 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
     5821 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagt cgaccatagc
     5881 caattcaata tggcgtatat ggactcatgc caattcaata tggtggatct ggacctgtgc
     5941 caattcaata tggcgtatat ggactcgtgc caattcaata tggtggatct ggaccccagc
     6001 caattcaata tggcggactt ggcaccatgc caattcaata tggcggactt ggcactgtgc
     6061 caactgggga ggggtctact tggcacggtg ccaagtttga ggaggggtct tggccctgtg
     6121 ccaagtccgc catattgaat tggcatggtg ccaataatgg cggccatatt ggctatatgc
     6181 caggatcaat atataggcaa tatccaatat ggccctatgc caatatggct attggccagg
     6241 ttcaatacta tgtattggcc ctatgccata tagtattcca tatatgggtt ttcctattga
     6301 cgtagatagc ccctcccaat gggcggtccc atataccata tatggggctt cctaataccg
     6361 cccatagcca ctcccccatt gacgtcaatg gtctctatat atggtctttc ctattgacgt
     6421 catatgggcg gtcctattga cgtatatggc gcctccccca ttgacgtcaa ttacggtaaa
     6481 tggcccgcct ggctcaatgc ccattgacgt caataggacc acccaccatt gacgtcaatg
     6541 ggatggctca ttgcccattc atatccgttc tcacgccccc tattgacgtc aatgacggta
     6601 aatggcccac ttggcagtac atcaatatct attaatagta acttggcaag tacattacta
     6661 ttggaaggac gccagggtac attggcagta ctcccattga cgtcaatggc ggtaaatggc
     6721 ccgcgatggc tgccaagtac atccccattg acgtcaatgg ggaggggcaa tgacgcaaat
     6781 gggcgttcca ttgacgtaaa tgggcggtag gcgtgcctaa tgggaggtct atataagcaa
     6841 tgctcgttta gggaac
//
