LOCUS       Exported                6498 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pCAGGS-TAL-NC2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6498)
  AUTHORS   Sakuma T, Hosoi S, Woltjen K, Suzuki KI, Kashiwagi K, Wada H, Ochiai
            H, Miyamoto T, Kawai N, Sasakura Y, Matsuura S, Okada Y, Kawahara A,
            Hayashi S, Yamamoto T
  TITLE     Efficient TALEN construction and evaluation methods for human cell 
            and animal applications.
  JOURNAL   Genes Cells. 2013 Feb 6. doi: 10.1111/gtc.12037.
  PUBMED    23388034
REFERENCE   2  (bases 1 to 6498)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6498
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1670
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1594..1616
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1678..1697
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1726..1735
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1747..1767
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1777..1800
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1801..1827
                     /codon_start=1
                     /product="epitope tag from the baculovirus Autographa 
                     californica GP64 envelope fusion protein"
                     /label=AcV5 tag
                     /translation="SWKDASGWS"
     CDS             complement(2191..2598)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLQGG
                     TRQCIARCMHCLHCGHAATFCNLHKLACVQL"
     primer_bind     2448..2470
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2462..2479
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2463..2479
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2489..2508
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2489..2507
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2555..2575)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2555..2573)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2594..2610)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2618..2634
                     /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(2642..2672)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2687..2708
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             2846..3433
                     /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(3522..3541)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3587..3642
                     /label=-beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3641..3660)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4001..4017)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4025..4041
                     /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(4049..4079)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4094..4115
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4174..4369
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4220..4355
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4282..4301
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    4375..4509
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4412..4431)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4466..4485
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4577..4594)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4748..5336)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4828..4847)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5507..6367)
                     /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     6130..6149
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6368..6472)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct
     1321 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg
     1381 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc
     1441 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt
     1501 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg
     1561 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg
     1621 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca
     1681 acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attccgccac catggcttcc
     1741 tcccctccaa agaaaaagag aaaggttgcg gccgctgact acaaggatga cgacgataaa
     1801 agttggaagg acgcaagtgg ttggtctaga atgcatgcgg ccccgcgacg gcgtgctgcg
     1861 caaccctccg acgcttcgcc ggccgcgcag gtggatctac gcacgctcgg ctacagtcag
     1921 cagcagcaag agaagatcaa accgaaggtg cgttcgacag tggcgcagca ccacgaggca
     1981 ctggtgggcc atgggtttac acacgcgcac atcgttgcgc tcagccaaca cccggcagcg
     2041 ttagggaccg tcgctgtcac gtatcagcac ataatcacgg cgttgccaga ggcgacacac
     2101 gaagacatcg ttggcgtcgg caaacagtgg tccggcgcac gcgccctgga ggccttgctc
     2161 acggatgcgg gggagttgag aggtccgccg ttacagttgg acacaggcca acttgtgaag
     2221 attgcaaaac gtggcggcgt gaccgcaatg gaggcagtgc atgcatcgcg caatgcactg
     2281 acgggtgccc ccctggagac gggcgccgct acagggcgcg tcccattcgc cattcaggct
     2341 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
     2401 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
     2461 ttgtaaaacg acggccagtg agcgcgcgta atacgactca ctatagggcg aattgggtac
     2521 cgggcccccc ctcgaggtcc tccagctttt gttcccttta gtgagggtta attgcgcgct
     2581 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     2641 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     2701 tcacattaat tgcgttgcgc tcactgcccg ctttccaccg gtcgtctcca acgaccacct
     2761 cgtcgccttg gcctgcctcg gcggacgtcc tgccatggat gcagtgaaaa agggattgcc
     2821 gcacgcgccg gaattgatca gatcccagct agtcaaaagt gaactggagg agaagaaatc
     2881 tgaacttcgt cataaattga aatatgtgcc tcatgaatat attgaattaa ttgaaattgc
     2941 cagaaattcc actcaggata gaattcttga aatgaaggta atggaatttt ttatgaaagt
     3001 ttatggatat agaggtaaac atttgggtgg atcaaggaaa ccggacggag caatttatac
     3061 tgtcggatct cctattgatt acggtgtgat cgtggatact aaagcttata gcggaggtta
     3121 taatctgcca attggccaag cagatgaaat gcaacgatat gtcgaagaaa atcaaacacg
     3181 aaacaaacat atcaacccta atgaatggtg gaaagtctat ccatcttctg taacggaatt
     3241 taagttttta tttgtgagtg gtcactttaa aggaaactac aaagctcagc ttacacgatt
     3301 aaatcatatc actaattgta atggagctgt tcttagtgta gaagagcttt taattggtgg
     3361 agaaatgatt aaagccggca cattaacctt agaggaagtg agaaggaaat ttaataacgg
     3421 cgagataaac ttttaatgga attcactcct caggtgcagg ctgcctatca gaaggtggtg
     3481 gctggtgtgg ccaatgccct ggctcacaaa taccactgag atctttttcc ctctgccaaa
     3541 aattatgggg acatcatgaa gccccttgag catctgactt ctggctaata aaggaaattt
     3601 attttcattg caatagtgtg ttggaatttt ttgtgtctct cactcggaag gacatatggg
     3661 agggcaaatc atttaaaaca tcagaatgag tatttggttt agagtttggc aacatatgcc
     3721 atatgctggc tgccatgaac aaaggtggct ataaagaggt catcagtata tgaaacagcc
     3781 ccctgctgtc cattccttat tccatagaaa agccttgact tgaggttaga ttttttttat
     3841 attttgtttt gtgttatttt tttctttaac atccctaaaa ttttccttac atgttttact
     3901 agccagattt ttcctcctct cctgactact cccagtcata gctgtccctc ttctcttatg
     3961 aagatccctc gacctgcagc ccaagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     4021 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     4081 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     4141 tccagtcggg aaacctgtcg tgccagcgga tccgcatctc aattagtcag caaccatagt
     4201 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     4261 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct
     4321 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctaacttg
     4381 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     4441 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     4501 gtctggatcc gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     4561 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     4621 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     4681 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     4741 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     4801 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     4861 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     4921 cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg gtgtaggtcg
     4981 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     5041 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     5101 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     5161 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     5221 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     5281 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     5341 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     5401 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     5461 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     5521 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     5581 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     5641 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     5701 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     5761 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     5821 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     5881 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     5941 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     6001 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     6061 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     6121 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     6181 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     6241 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     6301 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     6361 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     6421 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     6481 cccgaaaagt gccacctg
//
