LOCUS       pMK451_(BSD-P2A-        3765 bp DNA     circular SYN 25-OCT-2022
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 3765)
  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..3765
                     /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"
     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"
     primer_bind     1173..1198
                     /label=(3221) dTAG FOR
                     /note="color: black; sequence: 
                     CTGGTGCAGGCGCCggatcaGGAGTGCAGGTGGAAACCATC; added: 
                     2022-10-25"
     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: 2022-02-19"
     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     complement(1483..1531)
                     /label=(3005) dTAG-BamHI
                     /note="color: black; sequence: 
                     agtggatccGGCGCCTGCACCAGCGCCgccgccTTCCAGTTTTAGAAGC; added: 
                     2022-02-19"
     primer_bind     complement(1483..1505)
                     /label=(3006) dTAG-NheI
                     /note="color: black; sequence: 
                     ATAGCgctagcgccgccTTCCAGTTTTAGAAGC; added: 2022-02-19"
     CDS             1499..1522
                     /codon_start=1
                     /label=Linker
                     /note="color: #ff99cc"
                     /translation="GGGAGAGA"
     primer_bind     1509..1527
                     /label=(3221) dTAG FOR
                     /note="color: black; sequence: 
                     CTGGTGCAGGCGCCggatcaGGAGTGCAGGTGGAAACCATC; added: 
                     2022-10-25"
     primer_bind     complement(1519..1542)
                     /label=(3174) BromoTag REV
                     /note="color: black; sequence: gctctagaactagtggatccGGCG; 
                     added: 2022-10-25"
     promoter        complement(1577..1595)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
                     /note="color: #ffffff; direction: LEFT"
     primer_bind     complement(1616..1632)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
     protein_bind    1640..1656
                     /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(1664..1670,1671..1688,1689..1694))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="This reverse directional feature has 3 segments:
                        1: 1664 .. 1670 / #ffffff / -10
                        2: 1671 .. 1688 / #ffffff
                        3: 1689 .. 1694 / #ffffff / -35"
     protein_bind    1709..1730
                     /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(2018..2606)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     CDS             complement(join(2777..3568,3569..3637))
                     /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: 2777 .. 3568 / #ccffcc
                        2: 3569 .. 3637 / #ccffcc / signal sequence"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(3638..3742)
                     /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 cggcggcgct ggtgcaggcg ccggatccac tagttctaga gcggccgcca ccgcggtgga
     1561 gctccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat
     1621 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     1681 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     1741 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     1801 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     1861 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     1921 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     1981 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     2041 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     2101 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     2161 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     2221 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     2281 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     2341 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     2401 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     2461 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     2521 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     2581 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     2641 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     2701 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     2761 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     2821 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     2881 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     2941 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     3001 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     3061 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     3121 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     3181 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     3241 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     3301 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     3361 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     3421 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     3481 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     3541 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     3601 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     3661 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     3721 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccac
//
