LOCUS Exported 4690 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses sgRNA for targeting CrPEPC1 gene, paromomycin resistance. ACCESSION . VERSION . KEYWORDS pIKU801-CrPEPC1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4690) AUTHORS Kao PH, Ng IS TITLE CRISPRi mediated phosphoenolpyruvate carboxylase regulation to enhance the production of lipid in Chlamydomonas reinhardtii. JOURNAL Bioresour Technol. 2017 May 4. pii: S0960-8524(17)30619-3. doi: 10.1016/j.biortech.2017.04.111. PUBMED 28501380 REFERENCE 2 (bases 1 to 4690) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4690 /organism="synthetic DNA construct" /mol_type="other DNA" misc_RNA 21..96 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter complement(2130..2148) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(2169..2185) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2169..2185) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(2182..2204) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 2193..2209 /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(2217..2247) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2262..2283 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(2400..2417) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2571..3159) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2651..2670) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3330..4190) /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 3953..3972 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" ORIGIN 1 ggacgcggtg accacctacc gttttagagc tagaaatagc aagttaaaat aaggctagtc 61 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttttgcaaaa ttttccagat 121 cgatttcttc ttcctctgtt cttcggcgtt caatttctgg ggttttctct tcgttttctg 181 taactgaaac ctaaaatttg acctaaaaaa aatctcaaat aatatgattc agtggttttg 241 tacttttcag ttagttgagt tttgcagttc cgatgagata aaccaataga attcacgcgt 301 catatggagc tcgctgaggc ttgacatgat tggtgcgtat gtttgtatga agctacagga 361 ctgatttggc gggctatgag ggcgggggaa gctctggaag ggccgcgatg gggcgcgcgg 421 cgtccagaag gcgccatacg gcccgctggc ggcacccatc cggtataaaa gcccgcgacc 481 ccgaacggtg acctccactt tcagcgacaa acgagcactt atacatacgc gactattctg 541 ccgctataca taaccactca gctagcttaa gatcccatca agcttgcatg ccgggcgcgc 601 cagaaggagc gcagccaaac caggatgatg tttgatgggg tatttgagca cttgcaaccc 661 ttatccggaa gccccctggc ccacaaaggc taggcgccaa tgcaagcagt tcgcatgcag 721 cccctggagc ggtgccctcc tgataaaccg gccagggggc ctatgttctt tactttttta 781 caagagaagt cactcaacat cttaaaatgg ccaggtgagt cgacgagcaa gcccggcgga 841 tcaggcagcg tgcttgcaga tttgacttgc aacgcccgca ttgtgtcgac gaaggctttt 901 ggctcctctg tcgctgtctc aagcagcatc taaccctgcg tcgccgtttc catttgcagg 961 atggccactc cgccctcccc ggtgctgaag aatttcgaag catggacgat gcgttgcgtg 1021 cactgcgggg tcggtatccc ggttgtgagt gggttgttgt ggaggatggg gcctcggggg 1081 ctggtgttta tcggcttcgg ggtggtgggc gggagttgtt tgtcaaggtg gcagctctgg 1141 gggccggggt gggcttgttg ggtgaggctg agcggctggt gtggttggcg gaggtgggga 1201 ttcccgtacc tcgtgttgtg gagggtggtg gggacgagag ggtcgcctgg ttggtcaccg 1261 aagcggttcc ggggcgtccg gccagtgcgc ggtggccgcg ggagcagcgg ctggacgtgg 1321 cggtggcgct cgcggggctc gctcgttcgc tgcacgcgct ggactgggag cggtgtccgt 1381 tcgatcgcag tctcgcggtg acggtgccgc aggcggcccg tgctgtcgct gaagggagcg 1441 tcgacttgga ggatctggac gaggagcgga aggggtggtc gggggagcgg cttctcgccg 1501 agctggagcg gactcggcct gcggacgagg atctggcggt ttgccacggt gacctgtgcc 1561 cggacaacgt gctgctcgac cctcgtacct gcgaggtgac cgggctgatc gacgtggggc 1621 gggtcggccg tgcggaccgg cactccgatc tcgcgctggt gctgcgcgag ctggcccacg 1681 aggaggaccc gtggttcggg ccggagtgtt ccgcggcgtt cctgcgggag tacgggcgcg 1741 ggtgggatgg ggcggtatcg gaggaaaagc tggcgtttta ccggctgttg gacgagttct 1801 tctgagggac ctgatggtgt tggtggctgg gtagggttgc gtcgcgtggg tgacagcaca 1861 gtgtggacgt tgggatcccc gctccgtgta aatggaggcg ctcgttgatc tgagccttgc 1921 cccctgacga acggcggtgg atggaagata ctgctctcaa gtgctgaagc ggtagcttag 1981 ctccccgttt cgtgctgatc agtctttttc aacacgtaaa aagcggagga gttttgcaat 2041 tttgttggtt gtaacgatcc tccgttgatt ttggcctctt tctccatggg cgggctgggc 2101 gtatttgaag cgggtaccag cttttgttcc ctttagtgag ggttaatttc gagcttggcg 2161 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac 2221 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 2281 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 2341 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 2401 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 2461 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 2521 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 2581 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 2641 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 2701 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 2761 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 2821 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 2881 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 2941 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 3001 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 3061 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 3121 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 3181 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 3241 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 3301 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 3361 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 3421 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 3481 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 3541 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 3601 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 3661 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 3721 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 3781 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 3841 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 3901 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 3961 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 4021 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 4081 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 4141 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 4201 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggctg cagagatctc 4261 tcgagcgact tgccttccgc acaatacatc atttcttctt agcttttttt cttcttcttc 4321 gttcatacag tttttttttg tttatcagct tacattttct tgaaccgtag ctttcgtttt 4381 cttcttttta actttccatt cggagttttt gtatcttgtt tcatagtttg tcccaggatt 4441 agaatgatta ggcatcgaac cttcaagaat ttgattgaat aaaacatctt cattcttaag 4501 atatgaagat aatcttcaaa aggcccctgg gaatctgaaa gaagagaagc aggcccattt 4561 atatgggaaa gaacaatagt atttcttata taggcccatt taagttgaaa acaatcttca 4621 aaagtcccac atcgcttaga taagaaaacg aagctgagtt tatatacagc tagagtcgaa 4681 gtagtgattg //