LOCUS       Exported                3866 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with miRFP670.
ACCESSION   .
VERSION     .
KEYWORDS    pMaCTag-15
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3866)
  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 3866)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..3866
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             64..1008
                     /codon_start=1
                     /product="bright monomeric near-infrared fluorescent 
                     protein engineered from a bacterial phytochrome, with an 
                     emission maximum of 670 nm (Shcherbakova et al., 2016)"
                     /label=miRFP670
                     /label=ORF frame 1
                     /translation="VAGHASGSPAFGTASHSNCEHEEIHLAGSIQPHGALLVVSEHDHR
                     VIQASANAAEFLNLGSVLGVPLAEIDGDLLIKILPHLDPTAEGMPVAVRCRIGNPSTEY
                     CGLMHRPPEGGLIIELERAGPSIDLSGTLAPALERIRTAGSLRALCDDTVLLFQQCTGY
                     DRVMVYRFDEQGHGLVFSECHVPGLESYFGNRYPSSTVPQMARQLYVRQRVRVLVDVTY
                     QPVPLEPRLSPLTGRDLDMSGCFLRSMSPCHLQFLKDMGVRATLAVSLVVGGKLWGLVV
                     CHHYLPRFIRFELRAICKRLAERIATRITALES"
     polyA_signal    1015..1136
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1052..1071)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1106..1125
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    1143..1176
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     promoter        1183..1423
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     1183..1203
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1354..1373
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    1433..1466
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     primer_bind     complement(1503..1522)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1504..1522)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1609..1626)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1780..2368)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1860..1879)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2539..3399)
                     /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     3162..3181
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3400..3504)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3572..3590
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3628..3650)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3750..3769
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3850..2
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3850..1
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tctgtagcag gtcatgcctc tggcagcccc gcattcggga ccgcctctca ttcgaattgc
      121 gaacatgaag agatccacct cgccggctcg atccagccgc atggcgcgct tctggtcgtc
      181 agcgaacatg atcatcgcgt catccaggcc agcgccaacg ccgcggaatt tctgaatctc
      241 ggaagcgtac tcggcgttcc gctcgccgag atcgacggcg atctgttgat caagatcctg
      301 ccgcatctcg atcccaccgc cgaaggcatg ccggtcgcgg tgcgctgccg gatcggcaat
      361 ccctctacgg agtactgcgg tctgatgcat cggcctccgg aaggcgggct gatcatcgaa
      421 ctcgaacgtg ccggcccgtc gatcgatctg tcaggcacgc tggcgccggc gctggagcgg
      481 atccgcacgg cgggttcact gcgcgcgctg tgcgatgaca ccgtgctgct gtttcagcag
      541 tgcaccggct acgaccgggt gatggtgtat cgtttcgatg agcaaggcca cggcctggta
      601 ttctccgagt gccatgtgcc tgggctcgaa tcctatttcg gcaaccgcta tccgtcgtcg
      661 actgtcccgc agatggcgcg gcagctgtac gtgcggcagc gcgtccgcgt gctggtcgac
      721 gtcacctatc agccggtgcc gctggagccg cggctgtcgc cgctgaccgg gcgcgatctc
      781 gacatgtcgg gctgcttcct gcgctcgatg tcgccgtgcc atctgcagtt cctgaaggac
      841 atgggcgtgc gcgccaccct ggcggtgtcg ctggtggtcg gcggcaagct gtggggcctg
      901 gttgtctgtc accattatct gccgcgcttc atccgtttcg agctgcgggc gatctgcaaa
      961 cggctcgccg aaaggatcgc gacgcggatc accgcgcttg agagctgaac tagtaacttg
     1021 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     1081 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttagtcg
     1141 atataacttc gtatagcata cattatacga agttatgtcg aggagggcct atttcccatg
     1201 attccttcat atttgcatat acgatacaag gctgttagag agataattgg aattaatttg
     1261 actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg
     1321 tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga
     1381 aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac cgataacttc
     1441 gtatagcata cattatacga agttatggta ccgatgcagc tagcccgcgg atctgccggt
     1501 ctccctatag tgagtcgtat taatttcgat aagccaggtt aacctgcatt aatgaatcgg
     1561 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     1621 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     1681 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     1741 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     1801 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     1861 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     1921 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc
     1981 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     2041 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     2101 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     2161 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     2221 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     2281 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     2341 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     2401 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     2461 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     2521 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     2581 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     2641 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     2701 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     2761 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     2821 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     2881 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     2941 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     3001 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     3061 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     3121 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     3181 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     3241 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     3301 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     3361 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     3421 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     3481 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga
     3541 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct
     3601 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     3661 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     3721 tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
     3781 ccatatggac atattgtcgt tagaacgcgg ctacaattaa tacataacct tatgtatcat
     3841 acacatacga tttaggtgac actata
//
