LOCUS       sequence-44399-e        8058 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 8058)
  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 8058)
  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: MCHR1B_L (TAL 3008)
FEATURES             Location/Qualifiers
     source          1..8058
                     /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             3319..3906
                     /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             3922..3963
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3973..3990
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4013..4030)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4019..4243
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4289..4717
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4376..4395)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4586..4607
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4726..4746)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4731..5061
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4912..5047
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4974..4993
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        5109..5156
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5175..5573
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    5731..5852
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5768..5787)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5822..5841
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5901..5917)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5901..5917)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5914..5936)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5925..5941
                     /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(5949..5955,5956..5973,5974..5979))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5994..6015
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6132..6149)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6303..6891)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6383..6402)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7062..7853,7854..7922))
                     /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     7685..7704
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7923..8027)
                     /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 gaacaataat gggggaaagc aagccctgga aaccgtgcaa
     1501 aggttgttgc cggtcctttg tcaagaccac ggccttacac cggagcaagt cgtggccatt
     1561 gcaagcaatg ggggtggcaa acaggctctt gagacggttc agagacttct cccagttctc
     1621 tgtcaagccc acgggctgac tcccgatcaa gttgtagcga ttgcgaataa caatggaggg
     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 atcccacgac
     1981 ggtggcaaac aggctcttga gacggttcag agacttctcc cagttctctg tcaagcccac
     2041 gggctgactc ccgatcaagt tgtagcgatt gcgtccaacg gtggagggaa acaagcattg
     2101 gagactgtcc aacggctcct tcccgtgttg tgtcaagccc acggtttgac gcctgcacaa
     2161 gtggtcgcca tcgccaacaa caacggcggt aagcaggcgc tggaaacagt acagcgcctg
     2221 ctgcctgtac tgtgccagga tcatggactg accccagacc aggtagtcgc aatcgcgtca
     2281 aacggagggg gaaagcaagc cctggaaacc gtgcaaaggt tgttgccggt cctttgtcaa
     2341 gaccacggcc ttacaccgga gcaagtcgtg gccattgcaa gcaatggggg tggcaaacag
     2401 gctcttgaga cggttcagag acttctccca gttctctgtc aagcccacgg gctgactccc
     2461 gatcaagttg tagcgattgc gtcgaacatt ggagggaaac aagcattgga gactgtccaa
     2521 cggctccttc ccgtgttgtg tcaagcccac ggtttgacgc ctgcacaagt ggtcgccatc
     2581 gcctcgaatg gcggcggtaa gcaggcgctg gaaacagtac agcgcctgct gcctgtactg
     2641 tgccaggatc atggactgac cccagaccag gtagtcgcaa tcgcgtcaca tgacggggga
     2701 aagcaagccc tggaaaccgt gcaaaggttg ttgccggtcc tttgtcaaga ccacggcctt
     2761 acaccggagc aagtcgtggc cattgcaagc aacatcggtg gcaaacaggc tcttgagacg
     2821 gttcagagac ttctcccagt tctctgtcaa gcccacgggc tgactcccga tcaagttgta
     2881 gcgattgcgt cgcatgacgg agggaaacaa gcattggaga ctgtccaacg gctccttccc
     2941 gtgttgtgtc aagcccacgg tttgacgcct gcacaagtgg tcgccatcgc ctccaatatt
     3001 ggcggtaagc aggcgctgga aacagtacag cgcctgctgc ctgtactgtg ccaggatcat
     3061 ggactgacac ccgaacaggt ggtcgccatt gctaataata acggaggacg gccagccttg
     3121 gagtccatcg tagcccaatt gtccaggccc gatcccgcgt tggctgcgtt aacgaatgac
     3181 catctggtgg cgttggcatg tcttggtgga cgacccgcgc tcgatgcagt caaaaagggt
     3241 ctgcctcatg ctcccgcatt gatcaaaaga accaaccggc ggattcccga gagaacttcc
     3301 catcgagtcg cgggatccca actagtcaaa agtgaactgg aggagaagaa atctgaactt
     3361 cgtcataaat tgaaatatgt gcctcatgaa tatattgaat taattgaaat tgccagaaat
     3421 tccactcagg atagaattct tgaaatgaag gtaatggaat tttttatgaa agtttatgga
     3481 tatagaggta aacatttggg tggatcaagg aaaccggacg gagcaattta tactgtcgga
     3541 tctcctattg attacggtgt gatcgtggat actaaagctt atagcggagg ttataatctg
     3601 ccaattggcc aagcagatga aatgcaacga tatgtcgaag aaaatcaaac acgaaacaaa
     3661 catatcaacc ctaatgaatg gtggaaagtc tatccatctt ctgtaacgga atttaagttt
     3721 ttatttgtga gtggtcactt taaaggaaac tacaaagctc agcttacacg attaaatcat
     3781 atcactaatt gtaatggagc tgttcttagt gtagaagagc ttttaattgg tggagaaatg
     3841 attaaagccg gcacattaac cttagaggaa gtgagacgga aatttaataa cggcgagata
     3901 aacttttaag ggcccttcga aggtaagcct atccctaacc ctctcctcgg tctcgattct
     3961 acgcgtaccg gtcatcatca ccatcaccat tgagtttaaa cccgctgatc agcctcgact
     4021 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     4081 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     4141 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     4201 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     4261 accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg
     4321 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     4381 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     4441 cggggcatcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     4501 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     4561 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     4621 cctatctcgg tctattcttt tgatttataa gggattttgg ggatttcggc ctattggtta
     4681 aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt
     4741 tagggtgtgg aaagtcccca ggctccccag gcaggcagaa gtatgcaaag catgcatctc
     4801 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     4861 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     4921 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     4981 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     5041 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     5101 cagcacgtgt tgacaattaa tcatcggcat agtatatcgg catagtataa tacgacaagg
     5161 tgaggaacta aaccatggcc aagcctttgt ctcaagaaga atccaccctc attgaaagag
     5221 caacggctac aatcaacagc atccccatct ctgaagacta cagcgtcgcc agcgcagctc
     5281 tctctagcga cggccgcatc ttcactggtg tcaatgtata tcattttact gggggacctt
     5341 gtgcagaact cgtggtgctg ggcactgctg ctgctgcggc agctggcaac ctgacttgta
     5401 tcgtcgcgat cggaaatgag aacaggggca tcttgagccc ctgcggacgg tgtcgacagg
     5461 tgcttctcga tctgcatcct gggatcaaag cgatagtgaa ggacagtgat ggacagccga
     5521 cggcagttgg gattcgtgaa ttgctgccct ctggttatgt gtgggagggc taagcacttc
     5581 gtggccgagg agcaggactg acacgtgcta cgagatttcg attccaccgc cgccttctat
     5641 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     5701 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     5761 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     5821 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     5881 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     5941 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     6001 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     6061 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     6121 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     6181 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     6241 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     6301 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     6361 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     6421 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     6481 agcgtggcgc tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     6541 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     6601 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     6661 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     6721 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
     6781 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     6841 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     6901 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     6961 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     7021 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     7081 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     7141 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     7201 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     7261 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     7321 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     7381 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     7441 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     7501 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     7561 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     7621 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     7681 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     7741 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     7801 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     7861 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     7921 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     7981 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     8041 aaagtgccac ctgacgtc
//
