LOCUS       Exported                9434 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Protein expression plasmid for 2xNLS FnCpf1 in pET-21a backbone.
ACCESSION   .
VERSION     .
KEYWORDS    pET-21a_FnCpf1_2xNLS_protein_expression
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9434)
  AUTHORS   Liu P, Luk K, Shin M, Idrizi F, Kwok S, Roscoe B, Mintzer E, Suresh 
            S, Morrison K, Frazao JB, Bolukbasi MF, Ponnienselvan K, Luban J, 
            Zhu LJ, Lawson ND, Wolfe SA
  TITLE     Enhanced Cas12a editing in mammalian cells and zebrafish.
  JOURNAL   Nucleic Acids Res. 2019 Mar 20. pii: 5403491. doi: 
            10.1093/nar/gkz184.
  PUBMED    30892626
REFERENCE   2  (bases 1 to 9434)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9434
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     975..994
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1014..1091)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1297..1316
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1400..1419
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1400..1418
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1419..1443
                     /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)."
     RBS             1458..1480
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5388..5435
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5442..5522
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             5523..5543
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5565..5582
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5635..5653)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5649..5696
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5733..6188
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5820..5839)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6030..6051
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6214..6318
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6319..7179
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6537..6556)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7350..7938
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7839..7858
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8092..8109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    8124..8263
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(8189..8208)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8349..8371
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(8365..8556)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(9365..1013)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac
       61 agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt
      121 tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg
      181 tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg
      241 gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga
      301 acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg
      361 ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc
      421 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
      481 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
      541 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
      601 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
      661 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
      721 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
      781 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
      841 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
      901 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
      961 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
     1021 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
     1081 tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc
     1141 cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat
     1201 ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct
     1261 catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc
     1321 agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga
     1381 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat
     1441 tcccctctag aaataatttt gtttaacttt aagaaggaga tatacatatg agcatctacc
     1501 aggagttcgt caacaagtat tcactgagta agacactgcg gttcgagctg atcccacagg
     1561 gcaagacact ggagaacatc aaggcccgag gcctgattct ggacgatgag aagcgggcaa
     1621 aagactataa gaaagccaag cagatcattg ataaatacca ccagttcttt atcgaggaaa
     1681 ttctgagctc cgtgtgcatc agtgaggatc tgctgcagaa ttactcagac gtgtacttca
     1741 agctgaagaa gagcgacgat gacaacctgc agaaggactt caagtccgcc aaggacacca
     1801 tcaagaaaca gattagcgag tacatcaagg actccgaaaa gtttaaaaat ctgttcaacc
     1861 agaatctgat cgatgctaag aaaggccagg agtccgacct gatcctgtgg ctgaaacagt
     1921 ctaaggacaa tgggattgaa ctgttcaagg ctaactccga tatcactgat attgacgagg
     1981 cactggaaat catcaagagc ttcaagggat ggaccacata ctttaaaggc ttccacgaga
     2041 accgcaagaa cgtgtactcc agcaacgaca ttcctacctc catcatctac cgaatcgtcg
     2101 atgacaatct gccaaagttc ctggagaaca aggccaaata tgaatctctg aaggacaaag
     2161 ctcccgaggc aattaattac gaacagatca agaaagatct ggctgaggaa ctgacattcg
     2221 atatcgacta taagactagc gaggtgaacc agagggtctt ttccctggac gaggtgtttg
     2281 aaatcgccaa tttcaacaat tacctgaacc agtccggcat tactaaattc aataccatca
     2341 ttggcgggaa gtttgtgaac ggggagaata ccaagcgcaa gggaattaac gaatacatca
     2401 atctgtatag ccagcagatc aacgacaaaa ctctgaagaa atacaagatg tctgtgctgt
     2461 tcaaacagat cctgagtgat accgagtcca agtcttttgt cattgataaa ctggaagatg
     2521 actcagacgt ggtcactacc atgcagagct tttatgagca gatcgccgct ttcaagacag
     2581 tggaggaaaa atctattaag gaaactctga gtctgctgtt cgatgacctg aaagcccaga
     2641 agctggacct gagtaagatc tacttcaaaa acgataagag tctgacagac ctgtcacagc
     2701 aggtgtttga tgactattcc gtgattggga ccgccgtcct ggagtacatt acacagcaga
     2761 tcgctccaaa gaacctggat aatccctcta agaaagagca ggaactgatc gctaagaaaa
     2821 ccgagaaggc aaaatatctg agtctggaaa caattaagct ggcactggag gagttcaaca
     2881 agcacaggga tattgacaaa cagtgccgct ttgaggaaat cctggccaac ttcgcagcca
     2941 tccccatgat ttttgatgag atcgcccaga acaaagacaa tctggctcag atcagtatta
     3001 agtaccagaa ccagggcaag aaagacctgc tgcaggcttc agcagaagat gacgtgaaag
     3061 ccatcaagga tctgctggac cagaccaaca atctgctgca caagctgaaa atcttccata
     3121 ttagtcagtc agaggataag gctaatatcc tggataaaga cgaacacttc tacctggtgt
     3181 tcgaggaatg ttacttcgag ctggcaaaca ttgtccccct gtataacaag attaggaact
     3241 acatcacaca gaagccttac tctgacgaga agtttaaact gaacttcgaa aatagtaccc
     3301 tggccaacgg gtgggataag aacaaggagc ctgacaacac agctatcctg ttcatcaagg
     3361 atgacaagta ctatctggga gtgatgaata agaaaaacaa taagatcttc gatgacaaag
     3421 ccattaagga gaacaaaggg gaaggataca agaaaatcgt gtataagctg ctgcccggcg
     3481 caaataagat gctgcctaag gtgttcttca gcgccaagag tatcaaattc tacaacccat
     3541 ccgaggacat cctgcggatt agaaatcact caacacatac taagaacggg agcccccaga
     3601 agggatatga gaaatttgag ttcaacatcg aggattgcag gaagtttatt gacttctaca
     3661 agcagagcat ctccaaacac cctgaatgga aggattttgg cttccggttt tccgacacac
     3721 agagatataa ctctatcgac gagttctacc gcgaggtgga aaatcagggg tataagctga
     3781 cttttgagaa catttctgaa agttacatcg acagcgtggt caatcaggga aagctgtacc
     3841 tgttccagat ctataacaaa gatttttcag catacagcaa gggcagacca aacctgcata
     3901 cactgtactg gaaggccctg ttcgatgaga ggaatctgca ggacgtggtc tataaactga
     3961 acggagaggc cgaactgttt taccggaagc agtctattcc taagaaaatc actcacccag
     4021 ctaaggaggc catcgctaac aagaacaagg acaatcctaa gaaagagagc gtgttcgaat
     4081 acgatctgat taaggacaag cggttcaccg aagataagtt ctttttccat tgtccaatca
     4141 ccattaactt caagtcaagc ggcgctaaca agttcaacga cgagatcaat ctgctgctga
     4201 aggaaaaagc aaacgatgtg cacatcctga gcattgaccg aggagagcgg catctggcct
     4261 actataccct ggtggatggc aaagggaata tcattaagca ggatacattc aacatcattg
     4321 gcaatgaccg gatgaaaacc aactaccacg ataaactggc tgcaatcgag aaggatagag
     4381 actcagctag gaaggactgg aagaaaatca acaacattaa ggagatgaag gaaggctatc
     4441 tgagccaggt ggtccatgag attgcaaagc tggtcatcga atacaatgcc attgtggtgt
     4501 tcgaggatct gaacttcggc tttaagaggg ggcgctttaa ggtggaaaaa caggtctatc
     4561 agaagctgga gaaaatgctg atcgaaaagc tgaattacct ggtgtttaaa gataacgagt
     4621 tcgacaagac cggaggcgtc ctgagagcct accagctgac agctcccttt gaaactttca
     4681 agaaaatggg aaaacagaca ggcatcatct actatgtgcc agccggattc acttccaaga
     4741 tctgccccgt gaccggcttt gtcaaccagc tgtaccctaa atatgagtca gtgagcaagt
     4801 cccaggaatt tttcagcaag ttcgataaga tctgttataa tctggacaag gggtacttcg
     4861 agttttcctt cgattacaag aacttcggcg acaaggccgc taaggggaaa tggaccattg
     4921 cctccttcgg atctcgcctg atcaactttc gaaattccga taaaaaccac aattgggaca
     4981 ctagggaggt gtacccaacc aaggagctgg aaaagctgct gaaagactac tctatcgagt
     5041 atggacatgg cgaatgcatc aaggcagcca tctgtggcga gagtgataag aaatttttcg
     5101 ccaagctgac ctcagtgctg aatacaatcc tgcagatgcg gaactcaaag accgggacag
     5161 aactggacta tctgattagc cccgtggctg atgtcaacgg aaacttcttc gacagcagac
     5221 aggcacccaa aaatatgcct caggatgcag acgccaacgg ggcctaccac atcgggctga
     5281 agggactgat gctgctgggc cggatcaaga acaatcagga ggggaagaag ctgaacctgg
     5341 tcattaagaa cgaggaatac ttcgagtttg tccagaatag aaataacaaa aggccggcgg
     5401 ccacgaaaaa ggccggccag gcaaaaaaga aaaagggatc ctacccatac gatgttccag
     5461 attacgctta tccctacgac gtgcctgatt atgcataccc atatgatgtc cccgactatg
     5521 ccccaaagaa gaagcggaag gtcaagcttg cggccgcact cgagcaccac caccaccacc
     5581 actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
     5641 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     5701 aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     5761 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     5821 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     5881 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     5941 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     6001 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     6061 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     6121 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     6181 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     6241 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     6301 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     6361 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     6421 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     6481 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     6541 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     6601 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     6661 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     6721 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     6781 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     6841 gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc
     6901 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     6961 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     7021 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     7081 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     7141 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     7201 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     7261 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     7321 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     7381 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     7441 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     7501 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     7561 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     7621 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     7681 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     7741 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     7801 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     7861 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     7921 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     7981 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     8041 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     8101 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     8161 atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     8221 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     8281 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     8341 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     8401 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     8461 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     8521 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     8581 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     8641 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     8701 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     8761 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     8821 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     8881 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     8941 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     9001 gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc
     9061 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     9121 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     9181 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     9241 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     9301 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     9361 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     9421 ccaacgcgcg ggga
//
