LOCUS Exported 8652 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION PiggyBac cargo vector expressing rtTA, contains BsmBI sites for sgRNA cloning. ACCESSION . VERSION . KEYWORDS PB_rtTA_BsmBI SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8652) AUTHORS Schertzer MD, Thulson E, Braceros KCA, Lee DM, Hinkle ER, Murphy RM, Kim SO, Vitucci EM, Calabrese JM TITLE A piggyBac-based toolkit for inducible genome editing in mammalian cells. JOURNAL RNA. 2019 May 17. pii: rna.068932.118. doi: 10.1261/rna.068932.118. PUBMED 31101683 REFERENCE 2 (bases 1 to 8652) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8652 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..241 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1..21 /label=hU6-F /note="Human U6 promoter, forward primer" misc_RNA 266..341 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 372..1583 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 1420..1439 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 1642..2388 /codon_start=1 /product="improved tetracycline-controlled transactivator" /label=rtTA-Advanced /note="In the Tet-On(R) system, rtTA-Advanced binds to the Tet-responsive element and stimulates transcription only in the presence of tetracycline or doxycycline." /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA DALDDFDLDMLPADALDDFDLDMLPG" misc_feature 2426..2997 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(2591..2608) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 2818..2837 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 3013..3807 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(3067..3086) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3677..3696 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 3849..3970 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3886..3905) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3940..3959 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" repeat_region 4733..4767 /label=piggyBac left (5') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" primer_bind complement(5258..5274) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5258..5274) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5271..5293) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5282..5298 /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(5306..5336) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5351..5372 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5489..5506) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5660..6248) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5740..5759) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6419..7279) /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 7042..7061 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7280..7384) /gene="bla" /label=AmpR promoter rep_origin complement(7410..7865) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(7547..7568) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 7759..7778 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 7992..8014 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 8006..8023 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 8007..8023 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" repeat_region 8153..8215 /label=piggyBac right (3') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" ORIGIN 1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 181 atgcttatcg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 241 cgaaacaccg gagacggacg tctctgtttt agagctagaa atagcaagtt aaaataaggc 301 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttcc gcattaaccc 361 acccaagatc tggcctccgc gccgggtttt ggcgcctccc gcgggcgccc ccctcctcac 421 ggcgagcgct gccacgtcag acgaagggcg cagcgagcgt cctgatcctt ccgcccggac 481 gctcaggaca gcggcccgct gctcataaga ctcggcctta gaaccccagt atcagcagaa 541 ggacatttta ggacgggact tgggtgactc tagggcactg gttttctttc cagagagcgg 601 aacaggcgag gaaaagtagt cccttctcgg cgattctgcg gagggatctc cgtggggcgg 661 tgaacgccga tgattatata aggacgcgcc gggtgtggca cagctagttc cgtcgcagcc 721 gggatttggg tcgcggttct tgtttgtgga tcgctgtgat cgtcacttgg tgagtagcgg 781 gctgctgggc tggccggggc tttcgtggcc gccgggccgc tcggtgggac ggaagcgtgt 841 ggagagaccg ccaagggctg tagtctgggt ccgcgagcaa ggttgccctg aactgggggt 901 tggggggagc gcagcaaaat ggcggctgtt cccgagtctt gaatggaaga cgcttgtgag 961 gcgggctgtg aggtcgttga aacaaggtgg ggggcatggt gggcggcaag aacccaaggt 1021 cttgaggcct tcgctaatgc gggaaagctc ttattcgggt gagatgggct ggggcaccat 1081 ctggggaccc tgacgtgaag tttgtcactg actggagaac tcggtttgtc gtctgttgcg 1141 ggggcggcag ttatggcggt gccgttgggc agtgcacccg tacctttggg agcgcgcgcc 1201 ctcgtcgtgt cgtgacgtca cccgttctgt tggcttataa tgcagggtgg ggccacctgc 1261 cggtaggtgt gcggtaggct tttctccgtc gcaggacgca gggttcgggc ctagggtagg 1321 ctctcctgaa tcgacaggcg ccggacctct ggtgagggga gggataagtg aggcgtcagt 1381 ttctttggtc ggttttatgt acctatcttc ttaagtagct gaagctccgg ttttgaacta 1441 tgcgctcggg gttggcgagt gtgttttgtg aagtttttta ggcacctttt gaaatgtaat 1501 catttgggtc aatatgtaat tttcagtgtt agactagtaa attgtccgct aaattctggc 1561 cgtttttggc ttttttgtta gacgaagctt ggtaccgagc tcggatctcc accccgtacc 1621 ggtcctgcag tcgaattcac catgtctaga ctggacaaga gcaaagtcat aaacggagct 1681 ctggaattac tcaatggtgt cggtatcgaa ggcctgacga caaggaaact cgctcaaaag 1741 ctgggagttg agcagcctac cctgtactgg cacgtgaaga acaagcgggc cctgctcgat 1801 gccctgccaa tcgagatgct ggacaggcat catacccact tctgccccct ggaaggcgag 1861 tcatggcaag actttctgcg gaacaacgcc aagtcatacc gctgtgctct cctctcacat 1921 cgcgacgggg ctaaagtgca tctcggcacc cgcccaacag agaaacagta cgaaaccctg 1981 gaaaatcagc tcgcgttcct gtgtcagcaa ggcttctccc tggagaacgc actgtacgct 2041 ctgtccgccg tgggccactt tacactgggc tgcgtattgg aggaacagga gcatcaagta 2101 gcaaaagagg aaagagagac acctaccacc gattctatgc ccccacttct gagacaagca 2161 attgagctgt tcgaccggca gggagccgaa cctgccttcc ttttcggcct ggaactaatc 2221 atatgtggcc tggagaaaca gctaaagtgc gaaagcggcg ggccgaccga cgcccttgac 2281 gattttgact tagacatgct cccagccgat gcccttgacg actttgacct tgatatgctg 2341 cctgctgacg ctcttgacga ttttgacctt gacatgctcc ccgggtaact aagtaaggat 2401 ccgcggccgc actagaggaa ttccgcccct ctcccccccc cccctaacgt tactggccga 2461 agccgcttgg aataaggccg gtgtgcgttt gtctatatgt tattttccac catattgccg 2521 tcttttggca atgtgagggc ccggaaacct ggccctgtct tcttgacgag cattcctagg 2581 ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt 2641 cctctggaag cttcttgaag acaaacaacg tctgtagcga ccctttgcag gcagcggaac 2701 cccccacctg gcgacaggtg cctctgcggc caaaagccac gtgtataaga tacacctgca 2761 aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag agtcaaatgg 2821 ctctcctcaa gcgtattcaa caaggggctg aaggatgccc agaaggtacc ccattgtatg 2881 ggatctgatc tggggcctcg gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa 2941 cgtctaggcc ccccgaacca cggggacgtg gttttccttt gaaaaacacg atgataagct 3001 taccggtcca ccatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg 3061 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg 3121 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc 3181 ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct 3241 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa 3301 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg 3361 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa 3421 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat 3481 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg 3541 agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc 3601 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac 3661 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg 3721 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc 3781 tatcgccttc ttgacgagtt cttctgacaa tcaacctctg gattacaaaa tttgtggtta 3841 actaactaaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa 3901 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca 3961 atgtatctta tcatgtctgg aattgactca aatgatgtca attagtctat cagaagctca 4021 tctggtctcc cttccggggg acaagacatc cctgtttaat atttaaacag cagtgttccc 4081 aaactgggtt cttatatccc ttgctctggt caaccaggtt gcagggtttc ctgtcctcac 4141 aggaacgaag tccctaaaga aacagtggca gccaggttta gccccggaat tgactggatt 4201 ccttttttag ggcccattgg tatggctttt tccccgtatc cccccaggtg tctgcaggct 4261 caaagagcag cgagaagcgt tcagaggaaa gcgatcccgt gccaccttcc ccgtgcccgg 4321 gctgtccccg cacgctgccg gctcggggat gcggggggag cgccggaccg gagcggagcc 4381 ccgggcggct cgctgctgcc ccctagcggg ggagggacgt aattacatcc ctgggggctt 4441 tggggggggg ctgtccctga tatctataac aagaaaatat atatataata agttatcacg 4501 taagtagaac atgaaataac aatataatta tcgtatgagt taaatcttaa aagtcacgta 4561 aaagataatc atgcgtcatt ttgactcacg cggtcgttat agttcaaaat cagtgacact 4621 taccgcattg acaagcacgc ctcacgggag ctccaagcgg cgactgagat gtcctaaatg 4681 cacagcgacg gattcgcgct atttagaaag agagagcaat atttcaagaa tgcatgcgtc 4741 aattttacgc agactatctt tctagggtta atctagctgc atcaggatca tatcgtcggg 4801 tcttttttcc ggctcagtca tcgcccaagc tggcgctatc tgggcatcgg ggaggaagaa 4861 gcccgtgcct tttcccgcga ggttgaagcg gcatggaaag agtttgccga ggatgactgc 4921 tgctgcattg acgttgagcg aaaacgcacg tttaccatga tgattcggga aggtgtggcc 4981 atgcacgcct ttaacggtga actgttcgtt caggccacct gggataccag ttcgtcgcgg 5041 cttttccgga cacagttccg gatggtcagc ccgaagcgca tcagcaaccc gaacaatacc 5101 ggcgacagcc ggaactgccg tgccggtgtg cagattaatg acagcggtgc ggcgctggga 5161 tattacgtca gcgaggacgg gtatcctggc tggatgccgc agaaatggac atggataccc 5221 cgtgagttac ccggcgggcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa 5281 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct 5341 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc 5401 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 5461 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 5521 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag 5581 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 5641 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 5701 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 5761 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 5821 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt 5881 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 5941 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 6001 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 6061 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg 6121 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 6181 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 6241 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 6301 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 6361 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 6421 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 6481 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 6541 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc 6601 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 6661 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg 6721 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca 6781 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg 6841 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca 6901 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 6961 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 7021 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca 7081 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 7141 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg 7201 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 7261 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt 7321 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc 7381 cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt 7441 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat 7501 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa 7561 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg 7621 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa 7681 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc 7741 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag 7801 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg 7861 cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc 7921 ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac 7981 gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgagcgcg cctcgttcat 8041 tcacgttttt gaacccgtgg aggacgggca gactcgcggt gcaaatgtgt tttacagcgt 8101 gatggagcag atgaagatgc tcgacacgct gcagaacacg cagctagatt aaccctagaa 8161 agataatcat attgtgacgt acgttaaaga taatcatgtg taaaattgac gcatgtgttt 8221 tatcggtctg tatatcgagg tttatttatt aatttgaata gatattaagt tttattatat 8281 ttacacttac atactaataa taaattcaac aaacaattta tttatgttta tttatttatt 8341 aaaaaaaaca aaaactcaaa atttcttcta taaagtaaca aaacttttat gagggacagc 8401 ccccccccaa agcccccagg gatgtaatta cgtccctccc ccgctagggg gcagcagcga 8461 gccgcccggg gctccgctcc ggtccggcgc tccccccgca tccccgagcc ggcagcgtgc 8521 ggggacagcc cgggcacggg gaaggtggca cgggatcgct ttcctctgaa cgcttctcgc 8581 tgctctttga gcctgcagac acctgggggg atacggggaa aaggcctcca cggccccata 8641 aagatggtta at //