LOCUS Exported 6094 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Contains sgRNA which targets GFPmut3b. Under an aTc inducible promoter. Suicide vector inserts into slr0230 site of Synechocystis. Carries kanamycin resistance. Propogates in E. coli. ACCESSION . VERSION . KEYWORDS pMD19T-slr0230-PL31-sgRNANT1-KmR SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6094) AUTHORS Yao L, Cengic I, Anfelt J, Hudson EP TITLE Multiple Gene Repression in Cyanobacteria Using CRISPRi. JOURNAL ACS Synth Biol. 2015 Dec 28. PUBMED 26689101 REFERENCE 2 (bases 1 to 6094) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6094 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(29..51) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 151..170 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 364..386 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 378..395 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 379..395 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" misc_feature 1476..1497 /label=BioBrick prefix /note="BioBrick prefix for parts that do not start with ""ATG""" protein_bind 1498..1516 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" misc_RNA 1575..1650 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" terminator 1674..1745 /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 1761..1788 /label=T7Te terminator /note="phage T7 early transcription terminator" CDS complement(1839..2654) /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 2562..2581 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 2646..2665 /label=pENTR-R /note="pENTR vectors, reverse primer" misc_feature 2769..2789 /label=BioBrick suffix /note="universal suffix for all parts" primer_bind complement(3873..3889) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3873..3889) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3886..3908) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3897..3913 /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(3921..3951) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3966..3987 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4104..4121) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4275..4863) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4355..4374) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5034..5894) /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 5657..5676 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5895..5999) /gene="bla" /label=AmpR promoter primer_bind 6067..6085 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" ORIGIN 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt agaactcggt acgcgcggat 421 cttccagaga tttgccacct gacgtctaag aaaccataac cgttgtccct tttgtttcat 481 cgaccaacaa ccgccgggga aacgggagag tttgtattac aaagatgatg actatcgcct 541 gagttttctc tacggcagtt atctcacttt gactaatttg agtgccaagg aatggcaacg 601 catagaacag ttaaggcttt ctcccctcta cgtttccatc catgccacgg aagcatcggt 661 gcgggaaaga ttgttaaaaa atcatcgggc cggtcagatt cttgaccagt tggcttggtt 721 ccaagctaga agattacaga tccacgccca ggtggtggtt tgtcctggtg ttaatgatgg 781 taagcactta gagcaaactt tgagggactt agcccagttt caccaagggg aaaccccagc 841 ggtgatttca gtggcggtgg tgccggtggg tctaacccgt tttcgtcccc aggaagatga 901 gttaagcccc gttggtcaag ctaaagccac ggaggtaatt gcccaggttc aagctttgca 961 aaaggaattt gcccagcaac taggtggtaa ctttgtttgg ttggccgatg aatggtttct 1021 cattgcccgt caacctttgc caccggaaag ccattacgaa gactatcccc aaattggcaa 1081 cggggtaggg tctatccgtc aattcattaa ggagtttcaa caacaagctg cggaatttct 1141 ccccccggcg atcgccgaag ccaagacgtt gacctgggta gtgggcaatg ctgtagaaca 1201 agcgtttgag ctactggtgg aacagttaaa tcaggtaaag ggtttaacag ttaatttagc 1261 gcccttaaac agtgactatt ggggtcagga aattacggtg acgggactat tgacggggca 1321 ggatttaatc gctaaattgg caggcagaga tttaggggat ggtattctat tgcctgctct 1381 gatgttgaaa catgatgata ctcgcttctt ggatgacctc cgggtggccg atgtggctca 1441 aaagttgggc acaaccattt accctgtggc tgatggaatt cgcggccgct tctagagtcc 1501 ctatcagtga tagagattga catccctatc agtgatagat ataatgtgtg gtaccatcta 1561 attcaacaag aattgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat 1621 caacttgaaa aagtggcacc gagtcggtgc ttttttttac tagagccagg catcaaataa 1681 aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg 1741 ctctctacta gagtcacact ggctcacctt cgggtgggcc tttctgcgtt tatatactag 1801 aggctctgcc agtgttacaa ccaattaacc aattctgatt agaaaaactc atcgagcatc 1861 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt 1921 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat 1981 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa 2041 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa 2101 agcttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa 2161 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg 2221 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact 2281 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct 2341 gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc 2401 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta 2461 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc 2521 ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac 2581 ccatataaat cagcatccat gttggaattt aatcgcggcc tggagcaaga cgtttcccgt 2641 tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt 2701 catgatgata tatttttatc ttgtgcaatg taacatcaga gattttgaga cacaacgtgg 2761 ctttgttgta ctagtagcgg ccgctgcagc ggagtttaca taatctgaat cagtttctgg 2821 aattgacccc tctgatggcc accagggtca ccgatgccga tggcagtgct ttagttctaa 2881 tgagagaggg ggaaatatct atctttgaac aaatccatgg ccacaaaaat agtcttaagg 2941 gcaccattaa aggcgctttg cagaaggccc gccaggttaa cctcacattg gattcttcca 3001 ctgtcctgag ttattttgac cgtcaattgc gccaagaatt accggcgatc gcctgttata 3061 acacccccat tttgagtcac caagaggaag tgggccgctt atatatcttt agtcaagatc 3121 gtaactatag ctggactccc acccgtcgta agttactaca gctaatttct gatcaaaccg 3181 ctgtggcgat cgccaatagt gatttaaacc aaaagttgag atccagagaa agtcaggatc 3241 gggaattgga aattgcttca gaaattcaaa atcaactatt gccccgttgt tgtccccaaa 3301 ttaatggttt ggacattgct gcccagtgta aaaccgccag tcgggtaggt ggggattact 3361 acgatttcat tcctgccaac tatgaccaac tgcgccaggg ggattggtta tgtcgtaaca 3421 caagccattt gggggtgccc tggagtattg tcatcggcga tgtcatgggt aaaggagtac 3481 cggcgggtct aattatgacc atgactaggg ggatgctgcg ggcagaagtg ctgaaccgcc 3541 atagtccagc ccaaattctt aaccacctca accgggtaat gtatgccgac ctggaaaatt 3601 cccatcgctt tgtcaccttg ttttattctg aatataaccc cgaaacgagc atactttcct 3661 atagcaacgc cgcccataac cctcccctat tgtggcgggc cggcagtgac agcccccaat 3721 gtttaattcc cctggatacg gaaggagctt taatcggctt ggagtcggat tccaattacc 3781 gggatgccca gattcagctt gttcccccag tcgggaaacc tgtcgtgcca gaaatcgtcg 3841 aacggcaggc gtgcaaactt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 3901 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt 3961 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg 4021 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg 4081 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg 4141 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 4201 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 4261 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 4321 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 4381 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 4441 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 4501 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 4561 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 4621 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 4681 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg 4741 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 4801 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 4861 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 4921 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 4981 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa 5041 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc 5101 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct 5161 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca 5221 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt 5281 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt 5341 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc 5401 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc 5461 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt 5521 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact 5581 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc 5641 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt 5701 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg 5761 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct 5821 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa 5881 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt 5941 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatggg ggttccgcgc 6001 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc 6061 tataaaaata ggcgtatcac gaggcccttt cgtc //