LOCUS Exported 7046 bp ds-DNA circular SYN 12-NOV-2021 DEFINITION CRISPR donor plasmid to create GFP fusion proteins. ACCESSION . VERSION . KEYWORDS pSOX2-donor SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7046) TITLE ENCODE collection: CRISPR constructs REFERENCE 2 (bases 1 to 7046) AUTHORS . TITLE Direct Submission JOURNAL Exported Nov 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7046 /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" primer_bind 2633..2650 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 2634..2650 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" CDS 3633..3653 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 3666..3710 /codon_start=1 /product="affinity and epitope tag derived from pancreatic ribonuclease A" /label=S-Tag /translation="KETAAAKFERQHMDS" CDS 3750..3773 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" CDS 3801..4520 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFIGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQRNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLK EFVTAAGITLGMDELYK" primer_bind complement(3846..3867) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(4107..4126) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" misc_feature 4558..5143 /label=IRES2 /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(4724..4741) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 4951..4970 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 5225..6028 /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(5288..5307) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 5898..5917 /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 gacggccagt gaattcgagc tcggtaccca tgatggagac ggagctgaag ccgccgggcc 2701 cgcagcaaac ttcggggggc ggcggcggca actccaccgc ggcggcggcc ggcggctacc 2761 agaaaaacag cccggaccgc gtcaagcggc ccatgaatgc cttcatggtg tggtcccgcg 2821 ggctgcggcg caagatggcc caggagaacc ccaagatgca caactcggag atcagcaagc 2881 gcctgggcgc cgagtggaaa cttttgtcgg agacggagaa gcggccgttc atcgacgagg 2941 ctaagcggct gcgagcgctg cacatgaagg agcacccgga ttataaatac cggccccggc 3001 ggaaaaccaa gacgctcatg aagaaggata agtacacgct gcccggcggg ctgctggccc 3061 ccggcggcaa tagcatggcg agcggggtcg gggtgggcgc cggcctgggc gcgggcgtga 3121 accagcgcat ggacagttac gcgcacatga acggctggag caacggcagc tacagcatga 3181 tgcaggacca gctgggctac ccgcagcacc cgggcctcaa tgcgcacggc gcagcgcaga 3241 tgcagcccat gcaccgctac gacgtgagcg ccctgcagta caactccatg accagctcgc 3301 agacctacat gaacggctcg cccacctaca gcatgtccta ctcgcagcag ggcacccctg 3361 gcatggctct tggctccatg ggttcggtgg tcaagtccga ggccagctcc agcccccctg 3421 tggttacctc ttcctcccac tccagggcgc cctgccaggc cggggacctc cgggacatga 3481 tcagcatgta tctccccggc gccgaggtgc cggaacccgc cgcccccagc agacttcaca 3541 tgtcccagca ctaccagagc ggcccggtgc ccggcacggc cattaacggc acactgcccc 3601 tctcacacat ggggattcca actactgcaa gcgagaatct ttattttcag ggcgccgcca 3661 aattcaaaga aaccgctgct gctaaattcg aacgccagca catggacagc ggaggtggag 3721 gttcgagcgg tccctcgggt tcgtcgagcc tggaagttct gttccagggg cccctgtcgt 3781 cgagcggtcc ctcgggttcg atggtgagca aaggcgagga gctgttcatc ggggtggtgc 3841 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg 3901 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc 3961 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc 4021 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg 4081 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga 4141 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg 4201 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca 4261 tggccgacaa gcagaggaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg 4321 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg 4381 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg 4441 agaagcgcga tcacatggtc cttaaggagt tcgtgaccgc cgccgggatc actctcggca 4501 tggacgagct gtacaagtaa tgatctagag tcgagttaat taagaattcc gccccccccc 4561 ctctccctcc ccccccctaa cgttactggc cgaagccgct tggaataagg ccggtgtgcg 4621 tttgtctata tgttattttc caccatattg ccgtcttttg gcaatgtgag ggcccggaaa 4681 cctggccctg tcttcttgac gagcattcct aggggtcttt cccctctcgc caaaggaatg 4741 cagggtctgt tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg aagacaaaca 4801 acgtctgtag cgaccctttg caggcagcgg aaccccccac ctggcgacag gtgcctctgc 4861 ggccaaaagc cacgtgtata agatacacct gcaaaggcgg cacaacccca gtgccacgtt 4921 gtgagttgga tagttgtgga aagagtcaaa tggctctcct caagcgtatt caacaagggg 4981 ctgaaggatg cccagaaggt accccattgt atgggatctg atctggggcc tcggtgcaca 5041 tgctttacat gtgtttagtc gaggttaaaa aaacgtctag gccccccgaa ccacggggac 5101 gtggttttcc tttgaaaaac acgatgataa tatggccaca acctgttaca ttgcacaaga 5161 taaaaatata tcatcacgaa cagtaaaact gtctgcttac ataaacagta atacaagggg 5221 tgttatggga tcggccattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt 5281 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt 5341 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc 5401 cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc 5461 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga 5521 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat 5581 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca 5641 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga 5701 tgatctggac gaggagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc 5761 gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gacgcctgct tgccgaatat 5821 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga 5881 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg 5941 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt 6001 ctatcgcctt cttgacgagt tcttctgagt ctgagggccg gacagcgaac tggagggggg 6061 agaaattttc aaagaaaaac gagggaaatg ggaggggtgc aaaagaggag agtaagaaac 6121 agcatggaga aaacccggta cgctcaaaaa gaaaaaggaa aaaaaaaaat cccatcaccc 6181 acagcaaatg acagctgcaa aagagaacac caatcccatc cacactcacg caaaaaccgc 6241 gatgccgaca agaaaacttt tatgagagag atcctggact tctttttggg ggactatttt 6301 tgtacagaga aaaccggggg agggtgggga gggcggggga atggaccttg tatagatctg 6361 gaggaaagaa agctacgaaa aactttttaa aagttctagt ggtacggtag gagctttgca 6421 ggaagtttgc aaaagtcttt accaataata tttagagcta gtctccaagc gacgaaaaaa 6481 atgttttaat atttgcaagc aacttttgta cagtatttat cgagataaac atggcaatca 6541 aaatgtccat tgtttataag ctgagaattt gccaatattt ttcaaggaga ggcttcttgc 6601 tgaattttga ttctgcagct gaaatttagg acagttgcaa acgtgaaaag aagaaaatta 6661 ttcaaatttg gacattttaa ttgtttaaaa attgtacaaa aggaaaaaat tagaataagt 6721 actggcgaac catctctgtg gtcttgttta aaaagggcaa aagttttaga ctgtactaaa 6781 ttttataact tactgttaaa agcaaaaatg gccatgcagg ttgacgccgt tggtaattta 6841 taatagcttt tgttcgatcc caactttcca ttttgttcag ataaaaaaaa ccatgaaatt 6901 actgtgtttg aaatattttc ttatggtttg taatatttct gtaaatttat agtgatattt 6961 taaggttttc ccccctttat tttccgtagt tgtattttaa aagattcggc tctgtattat 7021 ttgaatcagt ctgccgagaa tccatg //