LOCUS Exported 5823 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION S. pyogenes Cas9 guide RNA expression in tilapia cells. ACCESSION . VERSION . KEYWORDS TU6m-gRNAscaffHygroR SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5823) AUTHORS Hamar J, Kultz D TITLE An efficient vector-based CRISPR/Cas9 system in an Oreochromis mossambicus cell line using endogenous promoters. JOURNAL Sci Rep. 2021 Apr 12;11(1):7854. doi: 10.1038/s41598-021-87068-3. PUBMED 33846462 REFERENCE 2 (bases 1 to 5823) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5823 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 1..20 /label=T7 /note="T7 promoter, forward primer" promoter 1..19 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 1099..2124 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK E" polyA_signal 2247..2368 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2284..2303) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2338..2357 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(3010..3030) /label=T3 /note="T3 promoter, forward primer" promoter complement(3010..3028) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(3049..3065) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3049..3065) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3062..3084) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3073..3089 /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(3097..3127) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3142..3163 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(3280..3297) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3451..4039) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3531..3550) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4210..5070) /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 4833..4852 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5071..5175) /gene="bla" /label=AmpR promoter rep_origin complement(5201..5656) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5338..5359) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 5550..5569 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5783..5805 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 5797..5814 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 5798..5814 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" ORIGIN 1 taatacgact cactataggg cgaattgggt accgggcccc ccctcgaggt cgacgttgca 61 gggtttcatt cggcggcgtg ctgacgtttt ttgcaggaat gtgtgtgatt tgcggggttt 121 tgggccgggg agggtgacgg gaaatgaggc cgcctcccat tgagtataaa acctcccagt 181 ccggcccctg tcttcctctt tctgttacct ggcttagggg cagcagcttt ggccgtgaaa 241 acccagaaac accggtgagt ttagctccca ttcttcacat tgagagggac ccggcctcgg 301 gcctttaaag gttttagtta aacgtgattt gttcaggcgt tagcatcctc tttgtgcttc 361 tttcttttac tcccaaactt tgctttttcg gttacgtaac ttgacactaa cctgtagatt 421 taattttcga gggaattaac cctaaaaaaa atagtaaagg tcgttctcgc ctgattgtat 481 ttaacatgat agttttttgt gaggtttgtg cgttagtgga tttactagat gctgtttttt 541 aaacttttga cgaaagattt tttacgaggg aggagaaaat gtagagtggg caacgggaac 601 aaaggagggc tgcaagaccg gagatccgaa gatgcttgaa tggcatttct tctctctcct 661 cactgaaaca actgcccgta tttatccggc aacaggtgct tgaagtccgg acaagatggc 721 tttgaatatt tggcgatctt tgtaacttta tccgacgtgg cccagtggag taacacgtgt 781 ctcggctggc ttgtggccga gtgaggccta gcagtagcag ccgcctggta gcggctaaag 841 cctagcgact gggtagccat tgagttttta acttagtata aattaaaaaa aaaacaatac 901 gttttagtag gctttctttt ttttttttaa acgcctttca ttaggcctat atgccgggat 961 ggtttcctaa tgtagctggg ctcaagatgc gatggtttag ttgtacatgc acatctgtta 1021 acgaagcttg acggctactt aatattaagc ctaacgcaat ttttatttac ttgcccacag 1081 aaactaaaac catgagatat gaaaaagcct gaactcaccg cgacgtctgt cgagaagttt 1141 ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct 1201 cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc 1261 gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt 1321 ccggaagtgc ttgacattgg ggaatttagc gagagcctga cctattgcat ctcccgccgt 1381 gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg 1441 gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc 1501 ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt 1561 gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc 1621 gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc 1681 gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc 1741 attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc 1801 tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg 1861 gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc 1921 tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac 1981 gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg 2041 gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc 2101 actcgtccga gggcaaagga atagagcggc cgcgactcta gatcataatc agccatacca 2161 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac 2221 ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat 2281 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg 2341 gtttgtccaa actcatcaat gtatcttaag gcgtaaattg taagcgttaa tattttgtta 2401 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc 2461 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt cccggtctga 2521 agtatactat gtgccgaatt tcccaccaag tggtaatgag caatgtttgt tattatggct 2581 cactattact tttgttctgc tgccttcagc cgtccaatca gaagcgtgtg agtaaacccg 2641 gtctgtgtaa attacccaca atgcctccgt gtgtctcact cacaggctca aagtgaaagc 2701 agagaatggg gaacagactc agactggcgc tctctgagcc tctgtgtctt cttacagatg 2761 tataaccgac ccagcagcac ccagacatgt ttctgctgct gtttcccatc agtttaaacg 2821 cagtttaagc gtcttccccc tgtaaccgtg actcacacag cttccaggac agagggcttt 2881 aaattgctcc cgggtccttg gagctatcga tgtgtgtcta gaaatagcaa gttaaaataa 2941 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tgagctccag 3001 cttttgttcc ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagctgtt 3061 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa 3121 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact 3181 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc 3241 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg 3301 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 3361 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag 3421 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 3481 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 3541 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 3601 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag 3661 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 3721 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 3781 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 3841 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt 3901 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 3961 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 4021 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 4081 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 4141 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 4201 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 4261 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 4321 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc 4381 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 4441 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 4501 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat 4561 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 4621 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 4681 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag 4741 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg 4801 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt 4861 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct 4921 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac 4981 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat 5041 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat 5101 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca 5161 aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt 5221 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa 5281 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca 5341 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc 5401 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg 5461 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg 5521 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg 5581 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg 5641 ccgctacagg gcgcgtccca ttcgccattc aggctgcgca actgttggga agggcgatcg 5701 gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta 5761 agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgagcgc 5821 gcg //