LOCUS Exported 7252 bp ds-DNA circular SYN 12-NOV-2021 DEFINITION CRISPR donor plasmid to create GFP fusion proteins. ACCESSION . VERSION . KEYWORDS pOTX2-donor SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7252) TITLE ENCODE collection: CRISPR constructs REFERENCE 2 (bases 1 to 7252) AUTHORS . TITLE Direct Submission JOURNAL Exported Nov 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7252 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(34..50) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(34..50) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(47..69) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 58..74 /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(82..112) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 127..148 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(265..282) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(436..1024) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(516..535) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(1195..2055) /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 1818..1837 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(2056..2160) /gene="bla" /label=AmpR promoter primer_bind 2228..2246 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(2284..2306) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 2406..2425 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 2619..2641 /label=M13/pUC Forward /note="In lacZ gene" CDS 3826..3846 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 3859..3903 /codon_start=1 /product="affinity and epitope tag derived from pancreatic ribonuclease A" /label=S-Tag /translation="KETAAAKFERQHMDS" CDS 3943..3966 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" CDS 3994..4713 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLK EFVTAAGITLGMDELYK" primer_bind complement(4039..4060) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(4300..4319) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" misc_feature 4751..5336 /label=IRES2 /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(4917..4934) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 5144..5163 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 5418..6221 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(5481..5500) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 6091..6110 /label=Neo-F /note="Neomycin resistance gene, forward primer" ORIGIN 1 gacctgcagg catgcaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg 61 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg 121 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc 181 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt 241 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct 301 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga 361 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 421 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 481 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 541 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 601 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 661 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 721 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 781 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 841 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct 901 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 961 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 1021 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg 1081 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta 1141 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca 1201 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc 1261 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc 1321 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc 1381 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat 1441 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt 1501 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc 1561 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag 1621 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt 1681 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac 1741 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg 1801 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat 1861 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc 1921 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc 1981 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa 2041 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg 2101 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg 2161 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac 2221 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga 2281 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg 2341 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa 2401 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca 2461 cagatgcgta aggagaaaat accgcatcag gcgccattcg ccattcaggc tgcgcaactg 2521 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg 2581 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac 2641 gacggccact gaattcgagc tcggtacccg agacagaagg gtgatggaac caaccaacct 2701 ctatattagc agttgcttcc tggaagatga actgtaaatg aatttaggct gttcctgtta 2761 gcctgtaaca acagaattgc tttctctcac ttctcatgta tttccctcca aaagacagag 2821 ttacattcta ttcctctgtg agagtgttcc aaaatatagc tgggtctatg tgtatgtgtg 2881 tgttagaggc gaaataagat aggaaagtga tgtggagagt ttttagctat atgatttggg 2941 ggattatctt aaatgttatt ctgggcaaga aataagacag tgtcaagtgt ttttagacag 3001 agcctcccca actttcttac aagtccagga gtttatatga gagtaccaca taataggtct 3061 tcagtggcag gggaaattgt gtgtttagct gatctgccca tgtaggatag atttataata 3121 cgggagccat tcttgtcctt aaggaactat caaaaccgag ttaaagaatt ttctttccct 3181 tccaaggtat ggtttaagaa tcgaagagct aagtgccgcc aacaacagca acaacagcag 3241 aatggaggtc aaaacaaagt gagacctgcc aaaaagaaga catctccagc tcgggaagtg 3301 agttcagaga gtggaacaag tggccaattc actcccccct ctagcacctc agtcccgacc 3361 attgccagca gcagtgctcc tgtgtctatc tggagcccag cttccatctc cccactgtca 3421 gatcccttgt ccacctcctc ttcctgcatg cagaggtcct atcccatgac ctatactcag 3481 gcttcaggtt atagtcaagg atatgctggc tcaacttcct actttggggg catggactgt 3541 ggatcatatt tgacccctat gcatcaccag cttcccggac caggggccac actcagtccc 3601 atgggtacca atgcagtcac cagccatctc aatcagtccc cagcttctct ttccacccag 3661 ggatatggag cttcaagctt gggttttaac tcaaccactg attgcttgga ttataaggac 3721 caaactgcct cctggaagct taacttcaat gctgactgct tggattataa agatcagaca 3781 tcctcgtgga aattccaggt tttggggatt ccaactactg caagcgagaa tctttatttt 3841 cagggcgccg ccaaattcaa agaaaccgct gctgctaaat tcgaacgcca gcacatggac 3901 agcggaggtg gaggttcgag cggtccctcg ggttcgtcga gcctggaagt tctgttccag 3961 gggcccctgt cgtcgagcgg tccctcgggt tcgatggtga gcaaaggcga ggagctgttc 4021 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc 4081 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc 4141 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg 4201 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg 4261 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc 4321 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc 4381 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac 4441 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc 4501 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc 4561 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc 4621 aaagacccca acgagaagcg cgatcacatg gtccttaagg agttcgtgac cgccgccggg 4681 atcactctcg gcatggacga gctgtacaag taatgatcta gagtcgagtt aattaagaat 4741 tccgcccccc cccctctccc tccccccccc taacgttact ggccgaagcc gcttggaata 4801 aggccggtgt gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt 4861 gagggcccgg aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct 4921 cgccaaagga atgcagggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc 4981 ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga 5041 caggtgcctc tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc 5101 ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt 5161 attcaacaag gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg 5221 gcctcggtgc acatgcttta catgtgttta gtcgaggtta aaaaaacgtc taggcccccc 5281 gaaccacggg gacgtggttt tcctttgaaa aacacgatga taatatggcc acaacctgtt 5341 acattgcaca agataaaaat atatcatcac gaacagtaaa actgtctgct tacataaaca 5401 gtaatacaag gggtgttatg ggatcggcca ttgaacaaga tggattgcac gcaggttctc 5461 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct 5521 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg 5581 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca 5641 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc 5701 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga 5761 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc 5821 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc 5881 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg 5941 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct 6001 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc 6061 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc 6121 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc 6181 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agtcagacct gtagaacctc 6241 tttttgtggg tgatttttaa atatactggg ctggacattc cagttttagc caggcattgg 6301 ttaaaagagt tggatgggat gatgctcaga ctcatctgat caaagttccg agaggcatag 6361 aaggaaaaac gaagggcctt agaggggcct acaaaccagc aacatgaaat ggacaaacca 6421 atctgcttaa gatcctgtca tagttttaga tcattggtta tcctgatttg caaagtgatc 6481 aaaagcattc tagccatgtg caaccaaaca ccaccaaaaa taaaatcaaa caaaactaag 6541 ttgtgaaggg agggagggaa ggtcatagcc ttcttaagca gaggtgttcc attgttttag 6601 ccaatccttg gttgaatctt aggaatgaac agtgtctcaa gctcattcac gtttcatgac 6661 caactggtag ttggcactga aaaaactttt cagggctgtg tgaattgtgt gactgattgt 6721 cctagatgca ctactttatt taaaaaataa tgttcataag gagtcaatat gtagtttaag 6781 agacaatcag tgtgtgtctt ataaatggta catctgtggt ttttaatctg tgctagactt 6841 caaaactgtg atctcctgtt attgtatgca accttgaact ccacctctgc aggggttctt 6901 ctgtgattaa ataggttata attataagca aaattcagag caactgagta ctgatctaaa 6961 aagattacct ttggctggag gtgagctgca ctgaaacttt acgacaaaat gtctctggac 7021 aaagagagtc agagaagaga agcaaaagga cactaattca tctgtaattt actgttggta 7081 agcctagcag taaagagaca ttggtcaatt gctctgaccc tgatgaatta ttaaactgag 7141 atcattgtcg tttatgcttg cagatgttaa atggaaaagt tatatatgca taaacctttt 7201 cttcctggat ttggcagata tgtataatta tattaaaatg gttctagcac aa //