LOCUS       40924_42624        7961 bp DNA     circular SYN 13-MAY-2021
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7961)
  AUTHORS   Sanjana NE, Cong L, Zhou Y, Cunniff MM, Feng G, Zhang F
  TITLE     A transcription activator-like effector toolbox for genome 
            engineering.
  JOURNAL   Nat Protoc. 2012 Jan 5;7(1):171-92. doi: 10.1038/nprot.2011.431.
  PUBMED    22222791
REFERENCE   2  (bases 1 to 7961)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7961)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/nprot.2011"; journalName: "Nat Protoc"; date: "2012-01-5-
            5"; volume: "7"; issue: "1"; pages: "171-92"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7961
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      86..674
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     828..845
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     944..960
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1416..1432
                     /label=lac operator
                     /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)."
     enhancer        1459..1838
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1839..2042
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        2087..2105
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2107..2116
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2116..2181
                     /codon_start=1
                     /label=3xFLAG
                     /note="three tandem FLAG(R) epitope tags, followed by an 
                     enterokinase cleavage site"
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2188..2208
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     promoter        2677..2707
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             2761..3417
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             3762..4064
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     CDS             4371..4958
                     /codon_start=1
                     /label=FokI cleavage domain
                     /note="nonspecific DNA cleavage domain of the FokI
                     endonuclease (Li et al., 1992)"
                     /translation="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKV
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQR
                     YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     polyA_signal    4982..5093
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        5216..5545
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             5594..6616
                     /codon_start=1
                     /label=HygR
                     /note="aminoglycoside phosphotransferase from E. coli"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    6749..6882
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6926..7783)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7784..7875)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7917..7936
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     7942..7961
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ctcatgacca aaatccctta acgtgagtta cgcgcgcgtc gttccactga gcgtcagacc
       61 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
      121 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
      181 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
      241 tgtagccgta gttagcccac cacttcaaga actctgtagc accgcctaca tacctcgctc
      301 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
      361 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
      421 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
      481 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
      541 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
      601 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
      661 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
      721 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
      781 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
      841 gcgaggaagc ggaaggcgag agtagggaac tgccaggcat caaactaagc agaaggcccc
      901 tgacggatgg cctttttgcg tttctacaaa ctctttctgt gttgtaaaac gacggccagt
      961 cttaagctcg ggccccctgg gcggttctga taacgagtaa tcgttaatcc gcaaataacg
     1021 taaaaacccg cttcggcggg tttttttatg gggggagttt agggaaagag catttgtcag
     1081 aatatttaag ggcgcctgtc actttgcttg atatatgaga attatttaac cttataaatg
     1141 agaaaaaagc aacgcacttt aaataagata cgttgctttt tcgattgatg aacacctata
     1201 attaaactat tcatctatta tttatgattt tttgtatata caatatttct agtttgttaa
     1261 agagaattaa gaaaataaat ctcgaaaata ataaagggaa aatcagtttt tgatatcaaa
     1321 attatacatg tcaacgataa tacaaaatat aatacaaact ataagatgtt atcagtattt
     1381 attatcattt agaataaatt ttgtgtcgcc cttaattgtg agcggataac aattacgagc
     1441 ttcatgcaca gtggcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg
     1501 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
     1561 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
     1621 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
     1681 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
     1741 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
     1801 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
     1861 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
     1921 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact
     1981 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag
     2041 ctctctggct aactagagaa cccactgctt actggcttat cgaaattaat acgactcact
     2101 ataggggcca ccatggacta taaggaccac gacggagact acaaggatca tgatattgat
     2161 tacaaagacg atgacgataa gatggcccca aagaagaagc ggaaggtcgg tatccacgga
     2221 gtcccagcag ccgtagattt gagaactttg ggatattcac agcagcagca ggaaaagatc
     2281 aagcccaaag tgaggtcgac agtcgcgcag catcacgaag cgctggtggg tcatgggttt
     2341 acacatgccc acatcgtagc cttgtcgcag caccctgcag cccttggcac ggtcgccgtc
     2401 aagtaccagg acatgattgc ggcgttgccg gaagccacac atgaggcgat cgtcggtgtg
     2461 gggaaacagt ggagcggagc ccgagcgctt gaggccctgt tgacggtcgc gggagagctg
     2521 agagggcctc cccttcagct ggacacgggc cagttgctga agatcgcgaa gcggggagga
     2581 gtcacggcgg tcgaggcggt gcacgcgtgg cgcaatgcgc tcacgggagc acccctcaac
     2641 ctgacagaga ccgcggccgc attaggcacc ccaggcttta cactttatgc ttccggctcg
     2701 tataatgtgt ggattttgag ttaggatccg tcgagatttt caggagctaa ggaagctaaa
     2761 atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca tcgtaaagaa
     2821 cattttgagg catttcagtc agttgctcaa tgtacctata accagaccgt tcagctggat
     2881 attacggcct ttttaaagac cgtaaagaaa aataagcaca agttttatcc ggcctttatt
     2941 cacattcttg cccgcctgat gaatgctcat ccggaattcc gtatggcaat gaaagacggt
     3001 gagctggtga tatgggatag tgttcaccct tgttacaccg ttttccatga gcaaactgaa
     3061 acgttttcat cgctctggag tgaataccac gacgatttcc ggcagtttct acacatatat
     3121 tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg gtttattgag
     3181 aatatgtttt tcgtctcagc caatccctgg gtgagtttca ccagttttga tttaaacgtg
     3241 gccaatatgg acaacttctt cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc
     3301 gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtttgtga tggcttccat
     3361 gtcggcagaa tgcttaatga attacaacag tactgcgatg agtggcaggg cggggcgtaa
     3421 agatctggat ccggcttact aaaagccaga taacagtatg cgtatttgcg cgctgatttt
     3481 tgcggtataa gaatatatac tgatatgtat acccgaagta tgtcaaaaag aggtatgcta
     3541 tgaagcagcg tattacagtg acagttgaca gcgacagcta tcagttgctc aaggcatata
     3601 tgatgtcaat atctccggtc tggtaagcac aaccatgcag aatgaagccc gtcgtctgcg
     3661 tgccgaacgc tggaaagcgg aaaatcagga agggatggct gaggtcgccc ggtttattga
     3721 aatgaacggc tcttttgctg acgagaacag gggctggtga aatgcagttt aaggtttaca
     3781 cctataaaag agagagccgt tatcgtctgt ttgtggatgt acagagtgat attattgaca
     3841 cgcccgggcg acggatggtg atccccctgg ccagtgcacg tctgctgtca gataaagtct
     3901 cccgtgaact ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg
     3961 atatggccag tgtgccggtc tccgttatcg gggaagaagt ggctgatctc agccaccgcg
     4021 aaaatgacat caaaaacgcc attaacctga tgttctgggg aatataaatg tcaggctccc
     4081 ttatacacag ccagtctgca ggtcgacggt ctcgactcac gcctgagcag gtagtggcta
     4141 ttgcatccaa catcgggggc agacccgcac tggagtcaat cgtggcccag ctttcgaggc
     4201 cggaccccgc gctggccgca ctcactaatg atcatcttgt agcgctggcc tgcctcggcg
     4261 gacgacccgc cttggatgcg gtgaagaagg ggctcccgca cgcgcctgca ttgattaagc
     4321 ggaccaacag aaggattccc gagaggacat cacatcgagt ggcaggttcc caactcgtga
     4381 agagtgaact tgaggagaaa aagtcggagc tgcggcacaa attgaaatac gtaccgcatg
     4441 aatacatcga acttatcgaa attgctagga actcgactca agacagaatc cttgagatga
     4501 aggtaatgga gttctttatg aaggtttatg gataccgagg gaagcatctc ggtggatcac
     4561 gaaaacccga cggagcaatc tatacggtgg ggagcccgat tgattacgga gtgatcgtcg
     4621 acacgaaagc ctacagcggt gggtacaatc ttcccatcgg gcaggcagat gagatgcaac
     4681 gttatgtcga agaaaatcag accaggaaca aacacatcaa tccaaatgag tggtggaaag
     4741 tgtatccttc atcagtgacc gagtttaagt ttttgtttgt ctctgggcat ttcaaaggca
     4801 actataaggc ccagctcaca cggttgaatc acattacgaa ctgcaatggt gcggttttgt
     4861 ccgtagagga actgctcatt ggtggagaaa tgatcaaagc gggaactctg acactggaag
     4921 aagtcagacg caagtttaac aatggcgaga tcaatttccg ctcataaaaa atcagcctcg
     4981 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     5041 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcacaacac
     5101 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     5161 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg
     5221 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     5281 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     5341 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     5401 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     5461 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt
     5521 ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc
     5581 tgatcagcac gtgatgaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat
     5641 cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc
     5701 tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg
     5761 tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga
     5821 agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca
     5881 gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc
     5941 ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt
     6001 cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga
     6061 tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca
     6121 ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca
     6181 cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga
     6241 ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag
     6301 gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct
     6361 tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca
     6421 gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat
     6481 cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt
     6541 ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg
     6601 tccgagggca aaggaatagc acgtgctacg agatttcgat tccaccgccg ccttctatga
     6661 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     6721 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     6781 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     6841 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     6901 gagcttggcg taatcatggt cattaccaat gcttaatcag tgaggcacct atctcagcga
     6961 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     7021 gggagggctt accatctggc cccagcgctg cgatgatacc gcgagaacca cgctcaccgg
     7081 ctccggattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     7141 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     7201 cgccagttaa tagtttgcgc aacgttgttg ccatcgctac aggcatcgtg gtgtcacgct
     7261 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     7321 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     7381 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     7441 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     7501 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     7561 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     7621 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     7681 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     7741 caaaaaaggg aataagggcg acacggaaat gttgaatact catattcttc ctttttcaat
     7801 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     7861 agaaaaataa acaaataggg gtcagtgtta caaccaatta accaattctg aacattatcg
     7921 cgagcccatt tatacctgaa tatggctcat aacacccctt g
//
