LOCUS Exported 8612 bp ds-DNA circular SYN 02-MAR-2018 DEFINITION -. ACCESSION - VERSION . KEYWORDS pTetR-P2A-BFPnls-sgRNA1-2XBroccoli-C3-6mer SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8612) AUTHORS UNKNOWN TITLE Direct Submission JOURNAL Exported Mar 2, 2018 from SnapGene 4.1.6 http://www.snapgene.com COMMENT ORIGDB|GenBank FEATURES Location/Qualifiers source 1..8612 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 1..507 /label=hPGK promoter /note="color: #66cc99; direction: RIGHT" primer_bind 434..451 /label=hPGK for seq for /note="color: black; sequence: cattctgcaagcctccgg" misc_feature 516..1088 /label=Rabbit beta-Globin Intron II (IVS) /note="color: #66cc99; direction: RIGHT" misc_feature 1172..1816 /label=TetR /note="color: #66cc99; direction: RIGHT" primer_bind 1326..1344 /label=TetR seq for /note="color: black; sequence: tcgacgccttagccattga" misc_feature 1826..1882 /label=P2A /note="color: #3366cc" misc_feature 1898..2608 /label=TagBFP2 /note="color: #66cc99; direction: RIGHT" primer_bind 2170..2189 /label=BFP seq for /note="color: black; sequence: cttcacatgggagagagtca" misc_feature 2198..2203 /label=(BbsI) /note="color: #3366cc" misc_feature 2483..2485 /label=V-to-I mutation /note="color: #3366cc" misc_feature 2609..2629 /label=NLS /note="color: #3366cc" misc_feature 2703..2716 /label=PPT /note="color: #3366cc" misc_feature 2724..2968 /label=SIN/3' LTR /note="color: #000000; direction: RIGHT" misc_feature 2724..2777 /label=Partial U3 /note="color: #3366cc" misc_feature 2749..2754 /label=(BbsI) /note="color: #3366cc" primer_bind 2799..2816 /label=LTR seq for /note="color: black; sequence: atctgagcctgggagctc" polyA_signal 3030..3160 /label=SV40 PolyA /note="color: #008000" primer_bind 3518..3535 /label=Middle seq for /note="color: black; sequence: tgaatggcgaatgggacg" promoter 4020..4118 /label=Bla Promoter /note="color: #800000; direction: RIGHT" misc_feature 4119..4979 /label=Amp /note="color: #66cc99; direction: RIGHT" primer_bind 4271..4290 /label=Amp resis seq for /note="color: black; sequence: cggtaagatccttgagagtt" primer_bind 5172..5191 /label=pUC Ori seq for /note="color: black; sequence: gtaatctgctgcttgcaaac" rep_origin 5180..5720 /direction=RIGHT /label=pUC Ori /note="color: #ffff00" misc_feature complement(join(5627..5643,6699,5644,6700..6795)) /label=Misc_Feature_1 /note="sgRNA cassette BfuAI sites [Split]" /note="This reverse directional feature has 4 segments: 1: 5627 .. 5643 / #a6acb3 2: 6699 .. 6699 / #a6acb3 3: 5644 .. 5644 / #a6acb3 4: 6700 .. 6795 / #a6acb3" primer_bind 6021..6042 /label=Misc seq primer /note="color: black; sequence: gcaattaatgtgagttagctca" misc_feature 6434..6614 /label=HIV 5' LTR /note="color: #000000; direction: RIGHT" primer_bind 6455..6472 /label=LTR seq for /note="color: black; sequence: atctgagcctgggagctc" misc_recomb 6681..6789 /label=Psi /note="color: #000080; direction: RIGHT" misc_recomb 6772..7099 /label=5' Gag /note="color: #000080; direction: RIGHT" primer_bind 6885..6903 /label=Gag seq for /note="color: black; sequence: cagggagctagaacgattc" misc_recomb 7290..7494 /label=REV /note="color: #000080; direction: RIGHT" primer_bind 7594..7614 /label=Before U6 seq for /note="color: black; sequence: acagatttggaatcacacgac" misc_feature 8029..8287 /label=U6 promoter /note="color: #66cc99; direction: RIGHT" primer_bind 8071..8091 /label=U6seq primer /note="color: black; sequence: gcatatacgatacaaggctgt" primer_bind 8256..8285 /label=GIBSON_for /note="color: black; sequence: ctttatatatcttgtggaaaggacgaaaca" misc_feature 8309..8394 /label=gRNA scaffold with AU flip and loop extension /note="color: #ffcc00" misc_feature 8395..8401 /label=terminator /note="color: #a6acb3" primer_bind complement(8407..8436) /label=GIBSON_rev /note="color: black; sequence: atgaatactgccatttgtctcgaggtcgag" ORIGIN 1 ttggggttgc gccttttcca aggcagccct gggtttgcgc agggacgcgg ctgctctggg 61 cgtggttccg ggaaacgcag cggcgccgac cctgggtctc gcacattctt cacgtccgtt 121 cgcagcgtca cccggatctt cgccgctacc cttgtgggcc ccccggcgac gcttcctgct 181 ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa 241 gccgcacgtc tcactagtac cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc 301 cgaccgcgat gggctgtggc caatagcggc tgctcagcag ggcgcgccga gagcagcggc 361 cgggaagggg cggtgcggga ggcggggtgt ggggcggtag tgtgggccct gttcctgccc 421 gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt 481 gaccgaatca ccgacctctc tccccagggg gatccgtgag tttggggacc cttgattgtt 541 ctttcttttt cgctattgta aaattcatgt tatatggagg gggcaaagtt ttcagggtgt 601 tgtttagaat gggaagatgt cccttgtatc accatggacc ctcatgataa ttttgtttct 661 ttcactttct actctgttga caaccattgt ctcctcttat tttcttttca ttttctgtaa 721 ctttttcgtt aaactttagc ttgcatttgt aacgaatttt taaattcact tttgtttatt 781 tgtcagattg taagtacttt ctctaatcac ttttttttca aggcaatcag ggtatattat 841 attgtacttc agcacagttt tagagaacaa ttgttataat taaatgataa ggtagaatat 901 ttctgcatat aaattctggc tggcgtggaa atattcttat tggtagaaac aactacatcc 961 tggtcatcat cctgcctttc tctttatggt tacaatgata tacactgttt gagatgagga 1021 taaaatactc tgagtccaaa ccgggcccct ctgctaacca tgttcatgcc ttcttctttt 1081 tcctacagct cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat 1141 tgtaatacga ctcactatag ggcgaattga tatgtctaga ttagataaaa gtaaagtgat 1201 taacagcgca ttagagctgc ttaatgaggt cggaatcgaa ggtttaacaa cccgtaaact 1261 cgcccagaag ctaggtgtag agcagcctac attgtattgg catgtaaaaa ataagcgggc 1321 tttgctcgac gccttagcca ttgagatgtt agataggcac catactcact tttgcccttt 1381 agaaggggaa agctggcaag attttttacg taataacgct aaaagtttta gatgtgcttt 1441 actaagtcat cgcgatggag caaaagtaca tttaggtaca cggcctacag aaaaacagta 1501 tgaaactctc gaaaatcaat tagccttttt atgccaacaa ggtttttcac tagagaatgc 1561 attatatgca ctcagcgctg tggggcattt tactttaggt tgcgtattgg aagatcaaga 1621 gcatcaagtc gctaaagaag aaagggaaac acctactact gatagtatgc cgccattatt 1681 acgacaagct atcgaattat ttgatcacca aggtgcagag ccagccttct tattcggcct 1741 tgaattgatc atatgcggat tagaaaaaca acttaaatgt gaaagtgggt ccgcgtacag 1801 cggctcccgg gagttcgcta gcggtgctac taacttcagc ctgctgaagc aggctggaga 1861 cgtggaggag aaccctggac ctggtagtgg aacgcgtatg gtgtctaagg gcgaagagct 1921 gattaaggag aacatgcaca tgaagctgta catggagggc accgtggaca accatcactt 1981 caagtgcaca tccgagggcg aaggcaagcc ctacgagggc acccagacca tgagaatcaa 2041 ggtggtcgag ggcggccctc tccccttcgc cttcgacatc ctggctacta gcttcctcta 2101 cggcagcaag accttcatca accacaccca gggcatcccc gacttcttca agcagtcctt 2161 ccctgagggc ttcacatggg agagagtcac cacatacgaa gatgggggcg tgctgaccgc 2221 tacccaggac accagcctcc aggacggctg cctcatctac aacgtcaaga tcagaggggt 2281 gaacttcaca tccaacggcc ctgtgatgca gaagaaaaca ctcggctggg aggccttcac 2341 cgagacgctg taccccgctg acggcggcct ggaaggcaga aacgacatgg ccctgaagct 2401 cgtgggcggg agccatctga tcgcaaacgc caagaccaca tatagatcca agaaacccgc 2461 taagaacctc aagatgcctg gcatctacta tgtggactac agactggaaa gaatcaagga 2521 ggccaacaac gaaacctacg tcgagcagca cgaggtggca gtggccagat actgcgacct 2581 ccctagcaaa ctggggcaca agcttaatcc aaagaaaaag cggaaagtgt gagtcgacct 2641 cgaggggggg cccggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc 2701 actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga agaaaagatc 2761 tgctttttgc ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg 2821 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag 2881 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag 2941 tgtggaaaat ctctagcagt agtagttcat gtcatcttat tattcagtat ttataacttg 3001 caaagaaatg aatatcagag agtgagagga acttgtttat tgcagcttat aatggttaca 3061 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt 3121 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gctctagcta tcccgcccct 3181 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg 3241 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa 3301 gtagtgagga ggcttttttg gaggcctagg gacgtaccca attcgcccta tagtgagtcg 3361 tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt 3421 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag 3481 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc 3541 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt 3601 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc 3661 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta 3721 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc 3781 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg 3841 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt 3901 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat 3961 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa 4021 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac 4081 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg 4141 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc 4201 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg 4261 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga 4321 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc 4381 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag 4441 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga 4501 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg 4561 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga 4621 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt 4681 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact 4741 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt 4801 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg 4861 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta 4921 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac 4981 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta 5041 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt 5101 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt 5161 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt 5221 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc 5281 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg 5341 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg 5401 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt 5461 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac 5521 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg 5581 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg 5641 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat 5701 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt 5761 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg 5821 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa 5881 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc 5941 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga 6001 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg 6061 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc 6121 acacaggaaa cagctatgac catgattacg ccaagcgcgc aattaaccct cactaaaggg 6181 aacaaaagct ggagctgcaa gcttaatgta gtcttatgca atactcttgt agtcttgcaa 6241 catggtaacg atgagttagc aacatgcctt acaaggagag aaaaagcacc gtgcatgccg 6301 attggtggaa gtaaggtggt acgatcgtgc cttattagga aggcaacaga cgggtctgac 6361 atggattgga cgaaccactg aattgccgca ttgcagagat attgtattta agtgcctagc 6421 tcgatacata aacgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta 6481 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg 6541 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg 6601 gaaaatctct agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag 6661 ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga 6721 ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag 6781 agcgtcagta ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca 6841 gggggaaaga aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga 6901 ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag 6961 ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca 7021 accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag 7081 atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcggccgc tgatcttcag 7141 acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt 7201 aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga 7261 aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac 7321 tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt 7381 gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac 7441 agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga 7501 tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc 7561 ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat 7621 ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc 7681 gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt 7741 gtggaattgg tttaacataa caaattggct gtggtatata aaattattca taatgatagt 7801 aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag 7861 gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg gacccgacag 7921 gcccgaagga atagaagaag aaggtggaga gagagacaga gacagatcca ttcgattagt 7981 gaacggatct cgacggtatc gatcacgaga ctagcctcga gcggccgccc ccttcaccga 8041 gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat 8101 aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa 8161 gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat 8221 gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg 8281 aaacaccgtt agggttaggg ttagggttgt ttaagagcta tgctggaaac agcatagcaa 8341 gtttaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt 8401 tgaattctcg acctcgagac aaatggcagt attcatccac aattttaaaa gaaaaggggg 8461 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac 8521 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag 8581 cagagatcca ctttggccgc ggctcgaggg gg //