LOCUS Exported 5507 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Fluorescent reporter for Sa gRNA (Bsa1 sites). ACCESSION . VERSION . KEYWORDS (401) pAAV CMV-ZsGreen U6-gRNA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5507) AUTHORS Backstrom JR, Sheng J, Wang MC, Bernardo-Colon A, Rex TS TITLE Optimization of S. aureus dCas9 and CRISPRi Elements for a Single Adeno-Associated Virus that Targets an Endogenous Gene. JOURNAL Mol Ther Methods Clin Dev. 2020 Sep 6;19:139-148. doi: 10.1016/j.omtm.2020.09.001. eCollection 2020 Dec 11. PUBMED 33024795 REFERENCE 2 (bases 1 to 5507) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5507 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..130 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" enhancer 210..513 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 514..716 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 667..687 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 713..737 /label=LNCX /note="Human CMV promoter, forward primer" regulatory 1336..1345 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1342..2037 /codon_start=1 /product="Zoanthus green fluorescent protein" /label=ZsGreen1 /note="mammalian codon-optimized" /translation="MAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKGKQAIN LCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFLFEDGAV CICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVPKQGILKG DVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKNQKWHLTEH AIASGSALP" primer_bind complement(2077..2094) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2083..2307 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 2412..2652 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2412..2432 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2583..2602 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 2681..2756 /label=Sa gRNA scaffold /note="guide RNA scaffold for the Staphylococcus aureus CRISPR/Cas9 system" repeat_region 2770..2910 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 2770..2899 /label=AAV2 ITR rep_origin 2985..3440 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3072..3091) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3282..3303 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3457..3476) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3576..3598 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(3636..3654) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 3722..3826 /gene="bla" /label=AmpR promoter CDS 3827..4687 /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 complement(4045..4064) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4858..5446 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5347..5366 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 121 aggggttcct gcggccgcac gcgtggagct agttattaat agtaatcaat tacggggtca 181 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct 241 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta 301 acgtcaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac 361 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt 421 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag 481 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat 541 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat 601 gggagtttgt tttgcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc 661 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt 721 tagtgaaccg tcagatcgcc tggagacgcc atccacgctg ttttgacctc catagaagac 781 accgggaccg atccagcctc cgcggattcg aatcccggcc gggaacggtg cattggaacg 841 cggattcccc gtgccaagag tgacgtaagt accgcctata gagtctatag gcccacaaaa 901 aatgctttct tcttttaata tacttttttg tttatcttat ttctaatact ttccctaatc 961 tctttctttc agggcaataa tgatacaatg tatcatgcct ctttgcacca ttctaaagaa 1021 taacagtgat aatttctggg ttaaggcaat agcaatattt ctgcatataa atatttctgc 1081 atataaattg taactgatgt aagaggtttc atattgctaa tagcagctac aatccagcta 1141 ccattctgct tttattttat ggttgggata aggctggatt attctgagtc caagctaggc 1201 ccttttgcta atcatgttca tacctcttat cttcctccca cagctcctgg gcaacgtgct 1261 ggtctgtgtg ctggcccatc actttggcaa agaattggga ttcgaacatc gattgaattc 1321 aggtactagt gatccgccac catggcccag tccaagcacg gcctgaccaa ggagatgacc 1381 atgaagtacc gcatggaggg ctgcgtggac ggccacaagt tcgtgatcac cggcgagggc 1441 atcggctacc ccttcaaggg caagcaggcc atcaacctgt gcgtggtgga gggcggcccc 1501 ttgcccttcg ccgaggacat cttgtccgcc gccttcatgt acggcaaccg cgtgttcacc 1561 gagtaccccc aggacatcgt cgactacttc aagaactcct gccccgccgg ctacacctgg 1621 gaccgctcct tcctgttcga ggacggcgcc gtgtgcatct gcaacgccga catcaccgtg 1681 agcgtggagg agaactgcat gtaccacgag tccaagttct acggcgtgaa cttccccgcc 1741 gacggccccg tgatgaagaa gatgaccgac aactgggagc cctcctgcga gaagatcatc 1801 cccgtgccca agcagggcat cttgaagggc gacgtgagca tgtacctgct gctgaaggac 1861 ggtggccgct tgcgctgcca gttcgacacc gtgtacaagg ccaagtccgt gccccgcaag 1921 atgcccgact ggcacttcat ccagcacaag ctgacccgcg aggaccgcag cgacgccaag 1981 aaccagaagt ggcacctgac cgagcacgcc atcgcctccg gctccgcctt gccctagctt 2041 ccacggtacc aagcttaagt ttaaaccgct gatcagcctc gactgtgcct tctagttgcc 2101 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca 2161 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta 2221 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc 2281 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaaggta accacgtgcg 2341 gaccgagcgg atccctagga ctgaacgaca tcttcgaagc tcagaaaatc gaatggcacg 2401 gatctggtac cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg 2461 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata 2521 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa 2581 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct 2641 tgtggaaagg acgaaacacc ggagaccacg gcaggtctca gttttagtac tctggaaaca 2701 gaatctacta aaacaaggca aaatgccgtg tttatctcgt caacttgttg gcgagatttt 2761 tgcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc 2821 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag 2881 tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc 2941 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg 3001 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc 3061 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc 3121 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct 3181 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac 3241 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac 3301 tggaacaaca ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat 3361 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa 3421 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata 3481 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct 3541 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt 3601 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata 3661 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt 3721 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag 3781 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca 3841 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc 3901 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat 3961 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc 4021 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg 4081 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc 4141 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat 4201 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga 4261 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc 4321 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc 4381 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt 4441 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc 4501 tggctggttt attgctgata aatctggagc cggtgagcgt ggaagccgcg gtatcattgc 4561 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca 4621 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca 4681 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt 4741 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta 4801 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg 4861 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc 4921 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag 4981 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa 5041 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 5101 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 5161 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 5221 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag 5281 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct 5341 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga 5401 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc 5461 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgt //