LOCUS Exported 4751 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Gateway destination vector only containing the Gateway cassette and pGEM-T Easy backbone. This can be used with Gateway-compatible Homology-Directed Repair.. ACCESSION . VERSION . KEYWORDS pGEM-gate SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4751) AUTHORS Mallett DR, Chang M, Cheng X, Bezanilla M TITLE Efficient and modular CRISPR-Cas9 vector system for Physcomitrella patens. JOURNAL Plant Direct. 2019 Sep 12;3(9):e00168. doi: 10.1002/pld3.168. eCollection 2019 Sep. PUBMED 31523744 REFERENCE 2 (bases 1 to 4751) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4751 /organism="synthetic DNA construct" /mol_type="other DNA" regulatory 33..42 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" regulatory 37..46 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind 77..201 /gene="mutant version of attR" /label=attR1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" promoter 226..256 /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" CDS 310..969 /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind complement(376..395) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" CDS 1311..1616 /codon_start=1 /gene="ccdB" /product="CcdB, a bacterial toxin that poisons DNA gyrase" /label=ccdB /note="Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains." /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind 1495..1514 /label=ccdB-fwd /note="ccdB gene, forward primer" protein_bind complement(1657..1781) /gene="mutant version of attR" /label=attR2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" promoter complement(1876..1894) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(1877..1894) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(1912..1928) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1912..1928) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(1925..1947) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 1936..1952 /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(1960..1990) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2005..2026 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(2143..2160) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2314..2902) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2394..2413) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3073..3933) /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 3696..3715 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(3934..4038) /gene="bla" /label=AmpR promoter rep_origin complement(4116..4571) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4253..4274) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 4465..4484 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4697..4719 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 4711..4728 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 4712..4728 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 4735..3 /label=T7 /note="T7 promoter, forward primer" promoter 4735..2 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" ORIGIN 1 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat 61 tcctaggtac gtaatcacaa gtttgtacaa aaaagctgaa cgagaaacgt aaaatgatat 121 aaatatcaat atattaaatt agattttgca taaaaaacag actacataat actgtaaaac 181 acaacatatc cagtcactat ggcggccgca ttaggcaccc caggctttac actttatgct 241 tccggctcgt ataatgtgtg gattttgagt taggatccgt cgagattttc aggagctaag 301 gaagctaaaa tggagaaaaa aatcactgga tataccaccg ttgatatatc ccaatggcat 361 cgtaaagaac attttgaggc atttcagtca gttgctcaat gtacctataa ccagaccgtt 421 cagctggata ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa gttttatccg 481 gcctttattc acattcttgc ccgcctgatg aatgctcatc cggaattccg tatggcaatg 541 aaagacggtg agctggtgat atgggatagt gttcaccctt gttacaccgt tttccatgag 601 caaactgaaa cgttttcatc gctctggagt gaataccacg acgatttccg gcagtttcta 661 cacatatatt cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg 721 tttattgaga atatgttttt cgtctcagcc aatccctggg tgagtttcac cagttttgat 781 ttaaacgtgg ccaatatgga caacttcttc gcccccgttt tcaccatggg caaatattat 841 acgcaaggcg acaaggtgct gatgccgctg gcgattcagg ttcatcatgc cgtctgtgat 901 ggcttccatg tcggcagaat gcttaatgaa ttacaacagt actgcgatga gtggcagggc 961 ggggcgtaaa cgcgtggatc cggcttacta aaagccagat aacagtatgc gtatttgcgc 1021 gctgattttt gcggtataag aatatatact gatatgtata cccgaagtat gtcaaaaaga 1081 ggtgtgctat gaagcagcgt attacagtga cagttgacag cgacagctat cagttgctca 1141 aggcatatat gatgtcaata tctccggtct ggtaagcaca accatgcaga atgaagcccg 1201 tcgtctgcgt gccgaacgct ggaaagcgga aaatcaggaa gggatggctg aggtcgcccg 1261 gtttattgaa atgaacggct cttttgctga cgagaacagg gactggtgaa atgcagttta 1321 aggtttacac ctataaaaga gagagccgtt atcgtctgtt tgtggatgta cagagtgata 1381 ttattgacac gcccgggcga cggatggtga tccccctggc cagtgcacgt ctgctgtcag 1441 ataaagtctc ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga 1501 tgaccaccga tatggccagt gtgccggtct ccgttatcgg ggaagaagtg gctgatctca 1561 gccaccgcga aaatgacatc aaaaacgcca ttaacctgat gttctgggga atataaatgt 1621 caggctccct tatacacagc cagtctgcag gtcgaccata gtgactggat atgttgtgtt 1681 ttacagtatt atgtagtctg ttttttatgc aaaatctaat ttaatatatt gatatttata 1741 tcattttacg tttctcgttc agctttcttg tacaaagtgg tgtacgtatt aattaaatca 1801 ctagtgaatt cgcggccgcc tgcaggtcga ccatatggga gagctcccaa cgcgttggat 1861 gcatagcttg agtattctat agtgtcacct aaatagcttg gcgtaatcat ggtcatagct 1921 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat 1981 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc 2041 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg 2101 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct 2161 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt 2221 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc 2281 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga 2341 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata 2401 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac 2461 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg 2521 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc 2581 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag 2641 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt 2701 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt 2761 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg 2821 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac 2881 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 2941 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac 3001 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac 3061 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt 3121 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt 3181 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt 3241 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc 3301 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa 3361 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg 3421 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt 3481 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc 3541 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt 3601 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg 3661 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac 3721 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc 3781 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt 3841 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg 3901 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag 3961 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa 4021 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgatgcgg tgtgaaatac 4081 cgcacagatg cgtaaggaga aaataccgca tcaggaaatt gtaagcgtta atattttgtt 4141 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg 4201 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg 4261 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta 4321 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg 4381 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa 4441 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct 4501 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct 4561 acagggcgcg tccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg 4621 gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg 4681 gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga attgtaatac 4741 gactcactat a //