LOCUS Exported 7402 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expresses AlRT-Cas1-Cas2 under pT7lac promoter, encodes AlCRISPR array 1, compatible with SENECA acquisition readout. ACCESSION . VERSION . KEYWORDS pFS_0369_pET30-RT-CRISPR_Azospirillum_lipoferum_4B_ARRAY_1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7402) AUTHORS Schmidt F, Cherepkova MY, Platt RJ TITLE Transcriptional recording by CRISPR spacer acquisition from RNA. JOURNAL Nature. 2018 Oct;562(7727):380-385. doi: 10.1038/s41586-018-0569-1. Epub 2018 Oct 3. PUBMED 30283135 REFERENCE 2 (bases 1 to 7402) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7402 /organism="synthetic DNA construct" /mol_type="other DNA" CDS complement(410..1225) /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind 1133..1152 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 1217..1236 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 1347..1935 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1836..1855 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2089..2106 /label=L4440 /note="L4440 vector, forward primer" misc_feature 2121..2260 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(2186..2205) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 2346..2368 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(2362..2553) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(3362..4444) /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" primer_bind 4406..4425 /label=LacI-R /note="LacI, reverse primer" promoter complement(4445..4522) /gene="lacI" /label=lacI promoter primer_bind 4728..4747 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 4835..4854 /label=T7 /note="T7 promoter, forward primer" promoter 4835..4853 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 4854..4878 /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 4893..4915 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" primer_bind complement(7315..7333) /label=T7 Term /note="T7 terminator, reverse primer" terminator 7329..7376 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" ORIGIN 1 ccaggtagag ctggtagacc attgaagata ttgggatatt gggctgttca ttgggtggtg 61 ttaaccactt ttccgggatt ttggagattt tatccacacc atgcgtggaa accgttggga 121 attttcccgt tgaatcaagc ggatgcgagg gataccgtcg cgacggcatc cgcgttcggg 181 agcggattga aacgtcccgc ggccgctatt cttttgattt ataagggatt ttgccgattt 241 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa 301 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 361 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact 421 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt 481 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa 541 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc 601 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg 661 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct 721 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga 781 gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc 841 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata 901 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac 961 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca 1021 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg 1081 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag 1141 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag 1201 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca 1261 gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac 1321 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc 1381 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca 1441 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta 1501 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct 1561 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg 1621 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc 1681 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta 1741 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg 1801 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt 1861 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg 1921 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggt cttttgctgg 1981 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc 2041 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg 2101 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt 2161 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt 2221 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc 2281 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac 2341 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca 2401 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc 2461 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc 2521 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct 2581 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga 2641 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg 2701 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg 2761 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg 2821 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagatca ttcatgttgt 2881 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga 2941 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag 3001 cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa 3061 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac 3121 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac 3181 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagatag tcataagtgc 3241 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa 3301 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg 3361 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca 3421 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg 3481 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt 3541 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat 3601 aacatgagct atcttcggta tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca 3661 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca 3721 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg 3781 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat 3841 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga 3901 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtacca tcttcatggg 3961 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat 4021 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca 4081 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc 4141 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg 4201 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca 4261 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca 4321 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc 4381 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt 4441 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg 4501 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt 4561 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca 4621 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg 4681 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg 4741 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg 4801 tagaggatcg agatcgatct cgatcccgcg aaattaatac gactcactat aggggaattg 4861 tgagcggata acaattcccc tctagaaata attttgttta actttaagaa ggagatatac 4921 atatgcctac ggcaccccct gataatgcag acctatttga agaagtgacc agattggaca 4981 cacttgagag agcttggggt agagtcttgc gaaatgctgg tgctgctggt ggtgacggtt 5041 tgactgtggg tcgttttgca gaagctgctc catctaggct tttggcatta catcgtactc 5101 ttagaatggg tgactatagg cctggtcccc ttagaagatt atctattcca aaaccagatg 5161 gtgctttaag accactggct atcccaccag tcactgatcg tgtcgctcaa actgctgctg 5221 ctcttgtatt gacgccactt ttggacggag aatttgagga cgcttcattt gggtatcgac 5281 ctggcagatc agttcctcaa gcagtagcca gggtagctag atggagagat caaggatacg 5341 attgggtcgt ggatgcagat attgagagat actttgagcg tgttccacat gatagacttc 5401 taatacgatt ggaaaggagt attggtgctg gtccacttac tgaattgatt gcagtgtggt 5461 tagagtcggg agccgaaaat ggtgttggtt tacctcaagg ttctccatta agtccattac 5521 tatctaattt gtatcttgat gacttagatg aagctcttga tggcagaggt ctacgtcttg 5581 ttaggtttgc agatgacttt gttctattgt gtagaagcag agaacgtgct gagagagctt 5641 tagatcacgc agctgctgta ttggaagaac acggtttgag actgaataga gataaaacca 5701 gaattgttcc ctttgatcaa ggatttaggt tcttgggtca cctatttgta agatccttgg 5761 ttttgccttc accaagacca gacgatggtg aagaaaccga aggtgacgca ttattgagag 5821 cactcgcaat tagagatgcc gccgctgaag cagaagaagc tgccgcagaa gaaagagata 5881 ggcaaagtcg tgctgctggg cttgatcctg cacttagaac gttacacgta caaacgccta 5941 gaaggagact agctttaagg aatgaggctt ttactgttgt tgagagggat gaccttggtg 6001 gtgaaagaga actcattgct attcatcatc atcatttgga tcgtattgat ctaggcccag 6061 aagcagatgc agacgctgaa gcattgagat gggcccttgc tacagatact gagttggcat 6121 ttgttgatgg ccacggtgct actcacggta gactaacgag gcctgaagct aggagagctg 6181 ctttgcattt agaccaagcc cgtcatgctc ttgatgaagg tttgcgttta gatctagcaa 6241 gaagaattgt agatggaaga ttgcgaaatc aaagagcact tcttagaaga ttgaatcgtt 6301 ccagaaaact tggagttgtc gaggacgctg tcctcgctat aaatgcacta cttagaagat 6361 tagatacagc tgctgatgtt gctgcattgt taggatttga aggtcaggga gctgctcgat 6421 actggccagc tttagctgcc caaattgaag gagagtggga atttgaagga agaaggcgta 6481 gaccaccccc tgaccctgta aatgctgtac tctcttacct ctcgggttta ttggagagag 6541 atgtagctgc tcttattgct aggcatgggc tacatccggg atttggagta ctacattcac 6601 cacaagatcg acatgatgca ggaatttatg atttgatgga agaatttaga gcccccttaa 6661 tggaaggcct ggctgttact ttgtttaata ggagaacgct caggccagat cactttagta 6721 gacgtgaaac gggtgaggga gaaattaaag gatgtcgtat cgaccccgat gctgttggag 6781 ctattattag ggcttacgag caatgggtcc gtcgtcctgt tgcatctcca agagatggta 6841 aaagaactac ttggagaggt cttataggat tccaagcaca agcattggct gcacacgtcc 6901 aaggaaggga accctataga gcttacgtga tggactatta gcgtacgtta actttaagaa 6961 ggagaactta agatgtctga aacaccaatg gttatggtgt tctgctacga tgtcgccaat 7021 gatagaagaa gaaggagagt ctcagcagta cttgaggagt ggggagttag ggtacaaaag 7081 tccgtatttg aagcaagact aactgagcct caagctagag ctttgttagc aagagctgga 7141 aaagaactgg gtccaagaga ttctcttaga atgtatgcac ttagcgcaca tggcctcgct 7201 cattctgctg ccctcggtgg ggctccagtt cccgagcaac aagacttttg gctcttgtaa 7261 ctcgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg 7321 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga 7381 aaggaggaac tatatccgga ta //