LOCUS Exported 3812 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Template for C-terminal PCR tagging of mammalian genes (without selection) with Halo. ACCESSION . VERSION . KEYWORDS pMaCTag-23 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3812) AUTHORS Fueller J, Herbst K, Meurer M, Gubicza K, Kurtulmus B, Knopf JD, Kirrmaier D, Buchmuller BC, Pereira G, Lemberg MK, Knop M TITLE CRISPR-Cas12a-assisted PCR tagging of mammalian genes. JOURNAL J Cell Biol. 2020 Jun 1;219(6). pii: 151766. doi: 10.1083/jcb.201910210. PUBMED 32406907 REFERENCE 2 (bases 1 to 3812) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3812 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..19 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 1..18 /label=SP6 /note="SP6 promoter, forward primer" CDS 81..968 /codon_start=1 /product="modified bacterial dehalogenase that forms covalent bonds with chloroalkane derivatives" /label=HaloTag(R) /translation="AEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSY VWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIH DWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIID QNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVE EYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLI GSEIARWLSTLEISG" polyA_signal 978..1099 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1015..1034) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 1069..1088 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" protein_bind 1106..1139 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 1146..1386 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1146..1166 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 1317..1336 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind 1396..1429 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(1466..1485) /label=T7 /note="T7 promoter, forward primer" promoter complement(1467..1485) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1572..1589) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(1743..2331) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1823..1842) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(2502..3362) /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 3125..3144 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(3363..3467) /gene="bla" /label=AmpR promoter primer_bind 3535..3553 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(3591..3613) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 3713..3732 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag 61 gtagtggcgg aggcggatcc gcagaaatcg gtactggctt tccattcgac ccccattatg 121 tggaagtcct gggcgagcgc atgcactacg tcgatgttgg tccgcgcgat ggcacccctg 181 tgctgttcct gcacggtaac ccgacctcct cctacgtgtg gcgcaacatc atcccgcatg 241 ttgcaccgac ccatcgctgc attgctccag acctgatcgg tatgggcaaa tccgacaaac 301 cagacctggg ttatttcttc gacgaccacg tccgcttcat ggatgccttc atcgaagccc 361 tgggtctgga agaggtcgtc ctggtcattc acgactgggg ctccgctctg ggtttccact 421 gggccaagcg caatccagag cgcgtcaaag gtattgcatt tatggagttc atccgcccta 481 tcccgacctg ggacgaatgg ccagaatttg cccgcgagac cttccaggcc ttccgcacca 541 ccgacgtcgg ccgcaagctg atcatcgatc agaacgtttt tatcgagggt acgctgccga 601 tgggtgtcgt ccgcccgctg actgaagtcg agatggacca ttaccgcgag ccgttcctga 661 atcctgttga ccgcgagcca ctgtggcgct tcccaaacga gctgccaatc gccggtgagc 721 cagcgaacat cgtcgcgctg gtcgaagaat acatggactg gctgcaccag tcccctgtcc 781 cgaagctgct gttctggggc accccaggcg ttctgatccc accggccgaa gccgctcgcc 841 tggccaaaag cctgcctaac tgcaaggctg tggacatcgg cccgggtctg aatctgctgc 901 aagaagacaa cccggacctg atcggcagcg agatcgcgcg ctggctgtcc acgctcgaga 961 tttccggctg aactagtaac ttgtttattg cagcttataa tggttacaaa taaagcaata 1021 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca 1081 aactcatcaa tgtatcttag tcgatataac ttcgtatagc atacattata cgaagttatg 1141 tcgaggaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta 1201 gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac 1261 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact 1321 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga 1381 aaggacgaaa caccgataac ttcgtatagc atacattata cgaagttatg gtaccgatgc 1441 agctagcccg cggatctgcc ggtctcccta tagtgagtcg tattaatttc gataagccag 1501 gttaacctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc 1561 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 1621 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 1681 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 1741 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 1801 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 1861 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 1921 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 1981 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 2041 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 2101 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 2161 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt 2221 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 2281 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 2341 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 2401 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 2461 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 2521 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 2581 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 2641 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 2701 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 2761 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 2821 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 2881 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 2941 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 3001 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 3061 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 3121 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 3181 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 3241 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 3301 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 3361 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 3421 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 3481 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg 3541 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac 3601 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc 3661 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca 3721 gagcagattg tactgagagt gcaccatatg gacatattgt cgttagaacg cggctacaat 3781 taatacataa ccttatgtat catacacata cg //