LOCUS Exported 2705 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION for expression of gRNA in E. coli. ACCESSION . VERSION . KEYWORDS pGRB SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 2705) AUTHORS Li Y, Lin Z, Huang C, Zhang Y, Wang Z, Tang YJ, Chen T, Zhao X TITLE Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. JOURNAL Metab Eng. 2015 Sep;31:13-21. doi: 10.1016/j.ymben.2015.06.006. Epub 2015 Jun 30. PUBMED 26141150 REFERENCE 2 (bases 1 to 2705) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..2705 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..35 /label=J23119(SpeI) promoter /note="bacterial promoter (Registry of Standard Biological Parts BBa_J23119) modified to end with an SpeI site" primer_bind complement(59..81) /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(176..493) /codon_start=1 /gene="lacZ fragment" /product="LacZ-alpha fragment of beta-galactosidase" /label=lacZ-alpha /translation="MITNSSSVPGDPLESTCRHASLALAVVLQRRDWENPGVTQLNRLA AHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICSDA A" primer_bind 181..200 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 394..416 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 408..425 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 409..425 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" misc_feature 429..485 /label=MCS /note="pUC18/19 multiple cloning site" primer_bind complement(494..510) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(494..510) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(507..529) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 518..534 /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(542..572) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 587..608 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(724..741) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(895..1483) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(975..994) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(1654..2514) /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 2277..2296 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(2515..2619) /gene="bla" /label=AmpR promoter primer_bind 2687..2705 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" ORIGIN 1 ttgacagcta gctcagtcct aggtataata ctagtttttt cggtgatgac ggtgaaaacc 61 tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca 121 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg 181 cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat 241 gcgtaaggag aaaataccgc atcaggcgcc attcgccatt caggctgcgc aactgttggg 301 aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg 361 caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg 421 ccagtgccaa gcttgcatgc ctgcaggtcg actctagagg atccccgggt accgagctcg 481 aattcgtaat catgtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac 541 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac 601 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcagttttag 661 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccc 721 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 781 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 841 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 901 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 961 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 1021 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 1081 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 1141 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 1201 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 1261 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 1321 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 1381 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 1441 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 1501 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 1561 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 1621 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 1681 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 1741 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgtgaccc 1801 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 1861 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 1921 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 1981 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 2041 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 2101 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 2161 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 2221 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 2281 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 2341 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 2401 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 2461 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 2521 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 2581 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 2641 acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac 2701 gaggc //