LOCUS       pMK453_(BSD-P2A-        4479 bp DNA     circular SYN 11-MAR-2022
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 4479)
  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..4479
                     /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"
     primer_bind     670..686
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; 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     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             1511..2218
                     /codon_start=1
                     /product="derivative of the monomeric red fluorescent 
                     protein mCherry"
                     /label=mCherry2
                     /note="mammalian codon-optimized"
                     /note="color: #ff0000"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFNWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQCRTMGWEASTERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVDIKLDILSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1717..1741)
                     /label=(1434) mCherry R2
                     /note="color: black; sequence: gtaggccttggagccgtacatgaac; 
                     added: 2022-03-11"
     primer_bind     complement(1795..1819)
                     /label=(1023)_mCherry2_R1
                     /note="color: black; sequence: GTTCATCACGCGCTCCCAATTGAAG; 
                     added: 2022-03-11"
     primer_bind     complement(2049..2071)
                     /label=(1867) mCherry_R
                     /note="color: black; sequence: cttgtaggtggtcttgacctcag; 
                     added: 2022-03-11"
     primer_bind     complement(2219..2245)
                     /label=(3005) dTAG-BamHI
                     /note="color: black; sequence: 
                     agtggatccGGCGCCTGCACCAGCGCCgccgccTTCCAGTTTTAGAAGC; added: 
                     2022-02-19"
     CDS             2219..2236
                     /codon_start=1
                     /label=Linker
                     /note="color: #ff99cc"
                     /translation="GAGAGA"
     CDS             2225..2236
                     /codon_start=1
                     /label=Linker-mAID-Clover
                     /note="color: #993366"
                     /translation="GAGA"
     misc_feature    2225..2234
                     
                     /note="color: #0000ff; direction: RIGHT"
     primer_bind     complement(2238..2254)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
     promoter        complement(2291..2309)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
                     /note="color: #ffffff; direction: LEFT"
     primer_bind     complement(2330..2346)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
     protein_bind    2354..2370
                     /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(2378..2384,2385..2402,2403..2408))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="This reverse directional feature has 3 segments:
                        1: 2378 .. 2384 / #ffffff / -10
                        2: 2385 .. 2402 / #ffffff
                        3: 2403 .. 2408 / #ffffff / -35"
     protein_bind    2423..2444
                     /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(2732..3320)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     CDS             complement(join(3491..4282,4283..4351))
                     /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: 3491 .. 4282 / #ccffcc
                        2: 4283 .. 4351 / #ccffcc / signal sequence"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4352..4456)
                     /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 ggataacatg gccatcatca aggagttcat
     1561 gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga
     1621 gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg
     1681 ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta
     1741 cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa
     1801 ttgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc
     1861 cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga
     1921 cggccccgta atgcagtgtc gtaccatggg ctgggaggcc tccactgagc ggatgtaccc
     1981 cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca
     2041 ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc
     2101 ctacaacgtc gacatcaagt tggacatcct ttcccacaac gaggactaca ccatcgtgga
     2161 acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaaggg
     2221 cgctggtgca ggcgccggat ccactagttc tagagcggcc gccaccgcgg tggagctcca
     2281 gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt
     2341 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     2401 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     2461 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     2521 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     2581 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     2641 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     2701 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     2761 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     2821 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     2881 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     2941 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     3001 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     3061 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     3121 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
     3181 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     3241 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     3301 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     3361 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     3421 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     3481 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     3541 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     3601 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     3661 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     3721 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     3781 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     3841 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     3901 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     3961 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     4021 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     4081 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     4141 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     4201 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     4261 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     4321 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     4381 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     4441 aaataggggt tccgcgcaca tttccccgaa aagtgccac
//
