LOCUS       pMK446_(BSD-P2A-        4566 bp DNA     circular SYN 18-FEB-2022
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 4566)
  AUTHORS   Molecular Function Laboratory
  TITLE     Direct Submission
  JOURNAL   Exported Feb 2, 2024 from SnapGene 7.1.1
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4566
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     rep_origin      4..459
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: RIGHT"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /note="color: #ffffff; direction: RIGHT"
     misc_feature    701..706
                     /label=Kozak seq
                     /note="color: #000000"
     CDS             707..1102
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /note="color: #ccffcc"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     960..982
                     /label=(1242) BSD seq1
                     /note="color: black; sequence: GCATCTTGAGCCCCTGCGGACGG"
     CDS             1115..1171
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /note="color: #ff0000
                     Cleavage site after base 1168"
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     1167..1197
                     /label=(2999) BglII-BromoTag
                     /note="color: black; sequence: 
                     GACCTagatctGTGAAGGACGTGCCCGACTC; added: 2022-02-18"
     CDS             1178..1564
                     /codon_start=1
                     /label=BromoTag
                     /note="color: #ff9900"
                     /translation="VKDVPDSQQHPAPEKSSKVSEQLKCCSGILKEMFAKKHAAYAWPF
                     YKPVDVEALGAHDYCDIIKHPMDMSTIKSKLEAREYRDAQEFGADVRLMFSNCYKYNPP
                     DHEVVAMARKLQDVFEMRFAKMPDE"
     modified_base   1343..1345
                     /label=L387
                     /mod_base="OTHER"
                     /note="color: #a6acb3"
     primer_bind     complement(1562..1583)
                     /label=(3000) BromoTag-BamHI
                     /note="color: black; sequence: 
                     agtggatccGGCGCCTGCACCAGCGCCGCCACCTCCGCCTCCGCCCTC; added: 
                     2022-02-18"
     primer_bind     complement(1564..1588)
                     /label=(3001) BromoTag-NheI
                     /note="color: black; sequence: 
                     ATAGCgctagcGCCACCTCCGCCTCCGCCC; added: 2022-02-18"
     primer_bind     1568..1609
                     /label=(1019)_CCC_FP_F
                     /note="color: black; sequence: 
                     CAAGAAGCTTaggtggaggaggttctggaggcggtggaagtggtggcggaggtagcggt
                     ggaggaggttctATGGTGAGCAAGGGCGAGGAG"
     CDS             complement(1587..2284)
                     /codon_start=1
                     /label=ORF frame 1
                     /note="color: #993366"
                     /translation="MCNPGGGHELQQDHVIALLVGVFAQLGLMAQVVVVGQQHGAVADG
                     GVLLVVVGELHAAVLNVVADLEVSLDAVLLLVGRDIDVVAVEVVLQLVPQDVAVLLEVD
                     ALQLDAVHQGVALELHLGAGLVGTVVLERDGALLDVAFGHGGLEEVVLLHVVGVAAEAG
                     HAVAEGGHEGGPGHGQLAGGADELQGQLAVGGIALALAADAELVAVYVAVQLDQDGHHP
                     GEQLLALAHH"
     CDS             join(1589..1591,1592..1594,1595..2305)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from GFP (Lam et al., 2012)"
                     /label=Clover
                     /note="This feature has 3 segments:
                        1: 1589 .. 1591 / #05fd14
                        2: 1592 .. 1594 / #05fd14 / 1a
                        3: 1595 .. 2305 / #05fd14"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     CDS             1589..2298
                     /codon_start=1
                     /label=ORF frame 2
                     /note="color: #993366"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDE"
     gene            1592..2298
                     /gene="EGFP (variant)"
                     /label=EGFP (variant)
                     /note="color: #a6acb3; direction: RIGHT"
     gene            1592..2298
                     /gene="ECFP (variant)"
                     /label=ECFP (variant)
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    complement(1634..1655)
                     /label=EGFP_N_primer
                     /note="color: #a6acb3; direction: LEFT"
     primer_bind     2136..2155
                     /label=(345) GFP seq 1
                     /note="color: black; sequence: ACCAGCAGAACACCCCCATC"
     primer_bind     complement(2192..2224)
                     /label=(1018)_mClover(A206K)_SDM_R
                     /note="color: black; sequence: 
                     ggggtctttgctcagCTTggactgatggctcag"
     primer_bind     2192..2224
                     /label=(1017)_mClover(A206K)_SDM_F
                     /note="color: black; sequence: 
                     ctgagccatcagtccAAGctgagcaaagacccc"
     misc_feature    2207..2209
                     /label=A206K
                     /note="color: #ff0000"
     misc_feature    2242..2263
                     /label=EGFP_C_primer
                     /note="color: #a6acb3"
     primer_bind     complement(2286..2305)
                     /label=(1020)_CCC_mClo_TAA_NotI_R
                     /note="color: black; sequence: 
                     ATGCTTGCGGCCGCTTACTTGTACAGCTCGTCCATGC"
     primer_bind     complement(2306..2332)
                     /label=(3000) BromoTag-BamHI
                     /note="color: black; sequence: 
                     agtggatccGGCGCCTGCACCAGCGCCGCCACCTCCGCCTCCGCCCTC; added: 
                     2022-02-18"
     CDS             2306..2323
                     /codon_start=1
                     /label=Linker
                     /note="color: #ff99cc"
                     /translation="GAGAGA"
     CDS             2312..2323
                     /codon_start=1
                     /label=Linker-mAID-Clover
                     /note="color: #993366"
                     /translation="GAGA"
     misc_feature    2312..2321
                     
                     /note="color: #0000ff; direction: RIGHT"
     promoter        complement(2378..2396)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
                     /note="color: #ffffff; direction: LEFT"
     primer_bind     complement(2417..2433)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
     protein_bind    2441..2457
                     /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)."
                     /note="color: #31849b"
     promoter        complement(join(2465..2471,2472..2489,2490..2495))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="This reverse directional feature has 3 segments:
                        1: 2465 .. 2471 / #ffffff / -10
                        2: 2472 .. 2489 / #ffffff
                        3: 2490 .. 2495 / #ffffff / -35"
     protein_bind    2510..2531
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
                     /note="color: #31849b"
     rep_origin      complement(2819..3407)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     CDS             complement(join(3578..4369,4370..4438))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                        1: 3578 .. 4369 / #ccffcc
                        2: 4370 .. 4438 / #ccffcc / signal sequence"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4439..4543)
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: LEFT"
ORIGIN
        1 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
       61 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
      121 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
      181 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
      241 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
      301 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
      361 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
      421 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg
      661 gccccccctc gaggtcgacg gtatcgataa gcttgatatc gccaccatgg ccaagccttt
      721 gtctcaagaa gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat
      781 ctctgaagac tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg
      841 tgtcaatgta tatcatttta ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc
      901 tgctgctgcg gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg
      961 catcttgagc ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa
     1021 agccatagtg aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc
     1081 ctctggttat gtgtgggagg gctcaggaag cggagctact aacttcagcc tgctgaagca
     1141 ggctggagac gtggaggaga accctggacc tagatctgtg aaggacgtgc ccgactctca
     1201 gcagcaccca gcaccagaga agagcagcaa ggtctcggag cagctcaagt gctgcagcgg
     1261 catcctcaag gagatgtttg ccaagaagca cgccgcctac gcctggccct tctacaagcc
     1321 tgtggacgtg gaggcactgg gcgcccacga ctactgtgac atcatcaagc accccatgga
     1381 catgagcaca atcaagtcta aactggaggc ccgtgagtac cgtgatgctc aggagtttgg
     1441 tgctgacgtc cgattgatgt tctccaactg ctataagtac aaccctcctg accatgaggt
     1501 ggtggccatg gcccgcaagc tccaggatgt gttcgaaatg cgctttgcca agatgccgga
     1561 cgagggcgga ggcggaggtg gcgctagcat ggtgagcaag ggcgaggagc tgttcaccgg
     1621 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtccg
     1681 cggcgagggc gagggcgatg ccaccaacgg caagctgacc ctgaagttca tctgcaccac
     1741 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ttcggctacg gcgtggcctg
     1801 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     1861 aggctacgtc caggagcgca ccatctcttt caaggacgac ggtacctaca agacccgcgc
     1921 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     1981 caaggaggac ggcaacatcc tggggcacaa gctggagtac aacttcaaca gccacaacgt
     2041 ctatatcacg gccgacaagc agaagaacgg catcaaggct aacttcaaga tccgccacaa
     2101 cgttgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     2161 cggccccgtg ctgctgcccg acaaccacta cctgagccat cagtccaagc tgagcaaaga
     2221 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggattac
     2281 acatggcatg gacgagctgt acaagggcgc tggtgcaggc gccggatcca ctagttctag
     2341 agcggccgcc accgcggtgg agctccagct tttgttccct ttagtgaggg ttaattgcgc
     2401 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     2461 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     2521 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     2581 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     2641 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     2701 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     2761 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     2821 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     2881 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     2941 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     3001 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     3061 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     3121 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     3181 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     3241 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
     3301 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     3361 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     3421 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     3481 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     3541 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     3601 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     3661 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     3721 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     3781 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     3841 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     3901 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     3961 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     4021 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     4081 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     4141 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     4201 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     4261 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     4321 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     4381 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     4441 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     4501 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     4561 tgccac
//
