LOCUS       pMK452_(BSD-P2A-        4488 bp DNA     circular SYN 19-FEB-2022
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 4488)
  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..4488
                     /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..1199
                     /label=(3004) BglII-dTAG
                     /note="color: black; sequence: 
                     GACCTagatctGGAGTGCAGGTGGAAACCATCT; added: 2022-02-19"
     CDS             1178..1498
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=dTAG (FKBP12F36V)
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /note="color: #008080"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     primer_bind     1178..1200
                     /label=(2997) dTAG FOR
                     /note="color: black; sequence: 
                     ggtctcgattctacgGGAGTGCAGGTGGAAACCATCTC; added: 2022-02-10"
     primer_bind     1178..1200
                     /label=(3007) SacI-dTAG
                     /note="color: black; sequence: 
                     aattggagctcCCCGggatccggtgcaggcgccGGAGTGCAGGTGGAAACCATCTC; 
                     added: 2022-02-19"
     primer_bind     complement(1476..1498)
                     /label=(2998) dTAG REV
                     /note="color: black; sequence: 
                     tccgatggatcctcaTTCCAGTTTTAGAAGCTCCACAT; added: 2022-02-10"
     primer_bind     complement(1476..1498)
                     /label=(3008) dTAG-MfeI
                     /note="color: black; sequence: 
                     agctaCAATTGttaTTCCAGTTTTAGAAGCTCCACAT; added: 2022-02-19"
     primer_bind     1480..1531
                     /label=(1019)_CCC_FP_F
                     /note="color: black; sequence: 
                     CAAGAAGCTTaggtggaggaggttctggaggcggtggaagtggtggcggaggtagcggt
                     ggaggaggttctATGGTGAGCAAGGGCGAGGAG"
     primer_bind     complement(1483..1510)
                     /label=(3006) dTAG-NheI
                     /note="color: black; sequence: 
                     ATAGCgctagcgccgccTTCCAGTTTTAGAAGC; added: 2022-02-19"
     primer_bind     complement(1483..1505)
                     /label=(3005) dTAG-BamHI
                     /note="color: black; sequence: 
                     agtggatccGGCGCCTGCACCAGCGCCgccgccTTCCAGTTTTAGAAGC; added: 
                     2022-02-19"
     CDS             complement(1509..2206)
                     /codon_start=1
                     /label=ORF frame 1
                     /note="color: #993366"
                     /translation="MCNPGGGHELQQDHVIALLVGVFAQLGLMAQVVVVGQQHGAVADG
                     GVLLVVVGELHAAVLNVVADLEVSLDAVLLLVGRDIDVVAVEVVLQLVPQDVAVLLEVD
                     ALQLDAVHQGVALELHLGAGLVGTVVLERDGALLDVAFGHGGLEEVVLLHVVGVAAEAG
                     HAVAEGGHEGGPGHGQLAGGADELQGQLAVGGIALALAADAELVAVYVAVQLDQDGHHP
                     GEQLLALAHH"
     CDS             join(1511..1513,1514..1516,1517..2227)
                     /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: 1511 .. 1513 / #05fd14
                        2: 1514 .. 1516 / #05fd14 / 1a
                        3: 1517 .. 2227 / #05fd14"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     CDS             1511..2220
                     /codon_start=1
                     /label=ORF frame 2
                     /note="color: #993366"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDE"
     gene            1514..2220
                     /gene="EGFP (variant)"
                     /label=EGFP (variant)
                     /note="color: #a6acb3; direction: RIGHT"
     gene            1514..2220
                     /gene="ECFP (variant)"
                     /label=ECFP (variant)
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    complement(1556..1577)
                     /label=EGFP_N_primer
                     /note="color: #a6acb3; direction: LEFT"
     primer_bind     2058..2077
                     /label=(345) GFP seq 1
                     /note="color: black; sequence: ACCAGCAGAACACCCCCATC"
     primer_bind     complement(2114..2146)
                     /label=(1018)_mClover(A206K)_SDM_R
                     /note="color: black; sequence: 
                     ggggtctttgctcagCTTggactgatggctcag"
     primer_bind     2114..2146
                     /label=(1017)_mClover(A206K)_SDM_F
                     /note="color: black; sequence: 
                     ctgagccatcagtccAAGctgagcaaagacccc"
     misc_feature    2129..2131
                     /label=A206K
                     /note="color: #ff0000"
     misc_feature    2164..2185
                     /label=EGFP_C_primer
                     /note="color: #a6acb3"
     primer_bind     complement(2208..2227)
                     /label=(1020)_CCC_mClo_TAA_NotI_R
                     /note="color: black; sequence: 
                     ATGCTTGCGGCCGCTTACTTGTACAGCTCGTCCATGC"
     primer_bind     complement(2228..2254)
                     /label=(3005) dTAG-BamHI
                     /note="color: black; sequence: 
                     agtggatccGGCGCCTGCACCAGCGCCgccgccTTCCAGTTTTAGAAGC; added: 
                     2022-02-19"
     CDS             2228..2245
                     /codon_start=1
                     /label=Linker
                     /note="color: #ff99cc"
                     /translation="GAGAGA"
     CDS             2234..2245
                     /codon_start=1
                     /label=Linker-mAID-Clover
                     /note="color: #993366"
                     /translation="GAGA"
     misc_feature    2234..2243
                     
                     /note="color: #0000ff; direction: RIGHT"
     promoter        complement(2300..2318)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
                     /note="color: #ffffff; direction: LEFT"
     primer_bind     complement(2339..2355)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
     protein_bind    2363..2379
                     /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(2387..2393,2394..2411,2412..2417))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="This reverse directional feature has 3 segments:
                        1: 2387 .. 2393 / #ffffff / -10
                        2: 2394 .. 2411 / #ffffff
                        3: 2412 .. 2417 / #ffffff / -35"
     protein_bind    2432..2453
                     /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(2741..3329)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     CDS             complement(join(3500..4291,4292..4360))
                     /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: 3500 .. 4291 / #ccffcc
                        2: 4292 .. 4360 / #ccffcc / signal sequence"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4361..4465)
                     /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 tagatctgga gtgcaggtgg aaaccatctc
     1201 cccaggagac gggcgcacct tccccaagcg cggccagacc tgcgtggtgc actacaccgg
     1261 gatgcttgaa gatggaaaga aagttgattc ctcccgggac agaaacaagc cctttaagtt
     1321 tatgctaggc aagcaggagg tgatccgagg ctgggaagaa ggggttgccc agatgagtgt
     1381 gggtcagaga gccaaactga ctatatctcc agattatgcc tatggtgcca ctgggcaccc
     1441 aggcatcatc ccaccacatg ccactctcgt cttcgatgtg gagcttctaa aactggaagg
     1501 cggcgctagc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     1561 cgagctggac ggcgacgtaa acggccacaa gttcagcgtc cgcggcgagg gcgagggcga
     1621 tgccaccaac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     1681 ctggcccacc ctcgtgacca ccttcggcta cggcgtggcc tgcttcagcc gctaccccga
     1741 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     1801 caccatctct ttcaaggacg acggtaccta caagacccgc gccgaggtga agttcgaggg
     1861 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     1921 cctggggcac aagctggagt acaacttcaa cagccacaac gtctatatca cggccgacaa
     1981 gcagaagaac ggcatcaagg ctaacttcaa gatccgccac aacgttgagg acggcagcgt
     2041 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     2101 cgacaaccac tacctgagcc atcagtccaa gctgagcaaa gaccccaacg agaagcgcga
     2161 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatt acacatggca tggacgagct
     2221 gtacaagggc gctggtgcag gcgccggatc cactagttct agagcggccg ccaccgcggt
     2281 ggagctccag cttttgttcc ctttagtgag ggttaattgc gcgcttggcg taatcatggt
     2341 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     2401 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     2461 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     2521 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     2581 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     2641 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     2701 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     2761 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     2821 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     2881 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     2941 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     3001 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     3061 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     3121 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     3181 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     3241 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     3301 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     3361 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     3421 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     3481 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     3541 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     3601 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     3661 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     3721 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     3781 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     3841 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     3901 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     3961 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     4021 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     4081 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     4141 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     4201 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     4261 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     4321 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     4381 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     4441 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccac
//
