LOCUS       sequence-44408-e        7956 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7956)
  AUTHORS   Sander JD, Cade L, Khayter C, Reyon D, Peterson RT, Joung JK, Yeh JR
  TITLE     Targeted gene disruption in somatic zebrafish cells using engineered
            TALENs.
  JOURNAL   Nat Biotechnol. 2011 Aug 5;29(8):697-8. doi: 10.1038/nbt.1934.
  PUBMED    21822241
REFERENCE   2  (bases 1 to 7956)
  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"; doi: "10.1038/nbt"; 
            journalName: "Nat Biotechnol"; date: "2011-08-5- 5"; volume: "29"; 
            issue: "8"; pages: "697-8"
COMMENT     Sequence Label: gabra5_R (TAL3015)
FEATURES             Location/Qualifiers
     source          1..7956
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             907..972
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             979..999
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3217..3804
                     /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"
     CDS             3820..3861
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3871..3888
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3911..3928)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3917..4141
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4187..4615
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4274..4293)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4484..4505
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4624..4644)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4629..4959
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4810..4945
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4872..4891
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        5007..5054
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5073..5471
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    5629..5750
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5666..5685)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5720..5739
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5799..5815)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5799..5815)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5812..5834)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5823..5839
                     /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(5847..5853,5854..5871,5872..5877))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5892..5913
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6030..6047)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6201..6789)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6281..6300)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6960..7751,7752..7820))
                     /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     7583..7602
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7821..7925)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 accatggact acaaagacca tgacggtgat tataaagatc atgacatcga ttacaaggat
      961 gacgatgaca agatggcccc caagaagaag aggaaggtgg gcattcaccg cggggtacct
     1021 atggtggact tgaggacact cggttattcg caacagcaac aggagaaaat caagcctaag
     1081 gtcaggagca ccgtcgcgca acaccacgag gcgcttgtgg ggcatggctt cactcatgcg
     1141 catattgtcg cgctttcaca gcaccctgcg gcgcttggga cggtggctgt caaataccaa
     1201 gatatgattg cggccctgcc cgaagccacg cacgaggcaa ttgtaggggt cggtaaacag
     1261 tggtcgggag cgcgagcact tgaggcgctg ctgactgtgg cgggtgagct tagggggcct
     1321 ccgctccagc tcgacaccgg gcagctgctg aagatcgcga agagaggggg agtaacagcg
     1381 gtagaggcag tgcacgcctg gcgcaatgcg ctcaccgggg cccccttgaa cctgacccca
     1441 gaccaggtag tcgcaatcgc gtcgaacatt gggggaaagc aagccctgga aaccgtgcaa
     1501 aggttgttgc cggtcctttg tcaagaccac ggccttacac cggagcaagt cgtggccatt
     1561 gcaaataata acggtggcaa acaggctctt gagacggttc agagacttct cccagttctc
     1621 tgtcaagccc acgggctgac tcccgatcaa gttgtagcga ttgcgtcgca tgacggaggg
     1681 aaacaagcat tggagactgt ccaacggctc cttcccgtgt tgtgtcaagc ccacggtttg
     1741 acgcctgcac aagtggtcgc catcgccagc catgatggcg gtaagcaggc gctggaaaca
     1801 gtacagcgcc tgctgcctgt actgtgccag gatcatggac tgaccccaga ccaggtagtc
     1861 gcaatcgcgt cgaacattgg gggaaagcaa gccctggaaa ccgtgcaaag gttgttgccg
     1921 gtcctttgtc aagaccacgg ccttacaccg gagcaagtcg tggccattgc aagcaatggg
     1981 ggtggcaaac aggctcttga gacggttcag agacttctcc cagttctctg tcaagcccac
     2041 gggctgactc ccgatcaagt tgtagcgatt gcgtcgcatg acggagggaa acaagcattg
     2101 gagactgtcc aacggctcct tcccgtgttg tgtcaagccc acggtttgac gcctgcacaa
     2161 gtggtcgcca tcgccagcca tgatggcggt aagcaggcgc tggaaacagt acagcgcctg
     2221 ctgcctgtac tgtgccagga tcatggactg accccagacc aggtagtcgc aatcgcgtcg
     2281 aacattgggg gaaagcaagc cctggaaacc gtgcaaaggt tgttgccggt cctttgtcaa
     2341 gaccacggcc ttacaccgga gcaagtcgtg gccattgcaa ataataacgg tggcaaacag
     2401 gctcttgaga cggttcagag acttctccca gttctctgtc aagcccacgg gctgactccc
     2461 gatcaagttg tagcgattgc gtcgcatgac ggagggaaac aagcattgga gactgtccaa
     2521 cggctccttc ccgtgttgtg tcaagcccac ggtttgacgc ctgcacaagt ggtcgccatc
     2581 gcctccaata ttggcggtaa gcaggcgctg gaaacagtac agcgcctgct gcctgtactg
     2641 tgccaggatc atggactgac cccagaccag gtagtcgcaa tcgcgtcgaa cattggggga
     2701 aagcaagccc tggaaaccgt gcaaaggttg ttgccggtcc tttgtcaaga ccacggcctt
     2761 acaccggagc aagtcgtggc cattgcaagc aatgggggtg gcaaacaggc tcttgagacg
     2821 gttcagagac ttctcccagt tctctgtcaa gcccacgggc tgactcccga tcaagttgta
     2881 gcgattgcgt cgcatgacgg agggaaacaa gcattggaga ctgtccaacg gctccttccc
     2941 gtgttgtgtc aagcccacgg tctgacaccc gaacaggtgg tcgccattgc ttcccacgac
     3001 ggaggacggc cagccttgga gtccatcgta gcccaattgt ccaggcccga tcccgcgttg
     3061 gctgcgttaa cgaatgacca tctggtggcg ttggcatgtc ttggtggacg acccgcgctc
     3121 gatgcagtca aaaagggtct gcctcatgct cccgcattga tcaaaagaac caaccggcgg
     3181 attcccgaga gaacttccca tcgagtcgcg ggatcccaac tagtcaaaag tgaactggag
     3241 gagaagaaat ctgaacttcg tcataaattg aaatatgtgc ctcatgaata tattgaatta
     3301 attgaaattg ccagaaattc cactcaggat agaattcttg aaatgaaggt aatggaattt
     3361 tttatgaaag tttatggata tagaggtaaa catttgggtg gatcaaggaa accggacgga
     3421 gcaatttata ctgtcggatc tcctattgat tacggtgtga tcgtggatac taaagcttat
     3481 agcggaggtt ataatctgcc aattggccaa gcagatgaaa tgcaacgata tgtcgaagaa
     3541 aatcaaacac gaaacaaaca tatcaaccct aatgaatggt ggaaagtcta tccatcttct
     3601 gtaacggaat ttaagttttt atttgtgagt ggtcacttta aaggaaacta caaagctcag
     3661 cttacacgat taaatcatat cactaattgt aatggagctg ttcttagtgt agaagagctt
     3721 ttaattggtg gagaaatgat taaagccggc acattaacct tagaggaagt gagacggaaa
     3781 tttaataacg gcgagataaa cttttaaggg cccttcgaag gtaagcctat ccctaaccct
     3841 ctcctcggtc tcgattctac gcgtaccggt catcatcacc atcaccattg agtttaaacc
     3901 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     3961 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     4021 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     4081 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     4141 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
     4201 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     4261 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     4321 ccccgtcaag ctctaaatcg gggcatccct ttagggttcc gatttagtgc tttacggcac
     4381 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     4441 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     4501 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgggg
     4561 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc
     4621 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccaggc aggcagaagt
     4681 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     4741 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     4801 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     4861 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
     4921 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
     4981 tccattttcg gatctgatca gcacgtgttg acaattaatc atcggcatag tatatcggca
     5041 tagtataata cgacaaggtg aggaactaaa ccatggccaa gcctttgtct caagaagaat
     5101 ccaccctcat tgaaagagca acggctacaa tcaacagcat ccccatctct gaagactaca
     5161 gcgtcgccag cgcagctctc tctagcgacg gccgcatctt cactggtgtc aatgtatatc
     5221 attttactgg gggaccttgt gcagaactcg tggtgctggg cactgctgct gctgcggcag
     5281 ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc ttgagcccct
     5341 gcggacggtg tcgacaggtg cttctcgatc tgcatcctgg gatcaaagcg atagtgaagg
     5401 acagtgatgg acagccgacg gcagttggga ttcgtgaatt gctgccctct ggttatgtgt
     5461 gggagggcta agcacttcgt ggccgaggag caggactgac acgtgctacg agatttcgat
     5521 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     5581 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
     5641 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     5701 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
     5761 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     5821 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     5881 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     5941 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     6001 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     6061 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     6121 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     6181 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     6241 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     6301 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     6361 gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag gtatctcagt
     6421 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     6481 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     6541 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     6601 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     6661 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     6721 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     6781 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     6841 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     6901 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     6961 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     7021 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     7081 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     7141 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     7201 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     7261 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     7321 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     7381 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     7441 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     7501 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     7561 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     7621 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     7681 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     7741 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     7801 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     7861 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     7921 ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
