LOCUS       sequence-147174-        7423 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  retroviral expression plasmid for selfcleaving E.coli biotin ligase 
            BirA-P2A-mCherry fusion protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7423)
  AUTHORS   Gabriel CH, Gross F, Karl M, Stephanowitz H, Hennig AF, Weber M, 
            Gryzik S, Bachmann I, Hecklau K, Wienands J, Schuchhardt J, Herzel 
            H, Radbruch A, Krause E, Baumgrass R
  TITLE     Identification of Novel Nuclear Factor of Activated T Cells 
            (NFAT)-Associated Proteins in T cells.
  JOURNAL   J Biol Chem. 2016 Sep 16. pii: jbc.M116.739326.
  PUBMED    27637333
REFERENCE   2  (bases 1 to 7423)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Biol 
            Chem. 2016 Sep 16. pii: jbc.M116.739326."
COMMENT     Sequence Label: pMSCV-BirA-P2A-mCherry
FEATURES             Location/Qualifiers
     source          1..7423
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             43..560
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    624..965
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             1032..1448
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1378..1400
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1416..1432
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      1469..1478
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1475..2440
                     /codon_start=1
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MEKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLR
                     DWGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKS
                     GDACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLR
                     KLGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQ
                     GWITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLII
                     GDKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             1475..2440
                     /codon_start=1
                     /gene="birA"
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MEKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLR
                     DWGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKS
                     GDACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLR
                     KLGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQ
                     GWITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLII
                     GDKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             2450..2506
                     /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."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2507..3217
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2652..2670)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2920..2939
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     LTR             3290..3805
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     primer_bind     complement(3974..3990)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3974..3990)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3987..4009)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3998..4014
                     /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)."
     promoter        complement(join(4022..4028,4029..4046,4047..4052))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4067..4088
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4205..4222)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4376..4964)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4456..4475)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5135..5926,5927..5995))
                     /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     5758..5777
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5996..6100)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6168..6186
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6224..6246)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6346..6365
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6388..6408)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6393..6722
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6573..6708
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6635..6654
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6770..6817
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     primer_bind     7011..7033
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7025..7042
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7026..7042
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7245..7264
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gataaaataa aagattttat ttagtctcca gaaaaagggg ggaatgaaag accccacctg
       61 taggtttggc aagctagctt aagtaacgcc attttgcaag gcatggaaaa tacataactg
      121 agaatagaga agttcagatc aaggttagga acagagagac agcagaatat gggccaaaca
      181 ggatatctgt ggtaagcagt tcctgccccg gctcagggcc aagaacagat ggtccccaga
      241 tgcggtcccg ccctcagcag tttctagaga accatcagat gtttccaggg tgccccaagg
      301 acctgaaaat gaccctgtgc cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt
      361 tcgcgcgctt ctgctccccg agctcaataa aagagcccac aacccctcac tcggcgcgcc
      421 agtcctccga tagactgcgt cgcccgggta cccgtattcc caataaagcc tcttgctgtt
      481 tgcatccgaa tcgtggactc gctgatcctt gggagggtct cctcagattg attgactgcc
      541 cacctcgggg gtctttcatt tggaggttcc accgagattt ggagacccct gcctagggac
      601 caccgacccc cccgccggga ggtaagctgg ccagcggtcg tttcgtgtct gtctctgtct
      661 ttgtgcgtgt ttgtgccggc atctaatgtt tgcgcctgcg tctgtactag ttagctaact
      721 agctctgtat ctggcggacc cgtggtggaa ctgacgagtt ctgaacaccc ggccgcaacc
      781 ctgggagacg tcccagggac tttgggggcc gtttttgtgg cccgacctga ggaagggagt
      841 cgatgtggaa tccgaccccg tcaggatatg tggttctggt aggagacgag aacctaaaac
      901 agttcccgcc tccgtctgaa tttttgcttt cggtttggaa ccgaagccgc gcgtcttgtc
      961 tgctgcagcg ctgcagcatc gttctgtgtt gtctctgtct gactgtgttt ctgtatttgt
     1021 ctgaaaatta gggccagact gttaccactc ccttaagttt gaccttaggt cactggaaag
     1081 atgtcgagcg gatcgctcac aaccagtcgg tagatgtcaa gaagagacgt tgggttacct
     1141 tctgctctgc agaatggcca acctttaacg tcggatggcc gcgagacggc acctttaacc
     1201 gagacctcat cacccaggtt aagatcaagg tcttttcacc tggcccgcat ggacacccag
     1261 accaggtccc ctacatcgtg acctgggaag ccttggcttt tgacccccct ccctgggtca
     1321 agccctttgt acaccctaag cctccgcctc ctcttcctcc atccgccccg tctctccccc
     1381 ttgaacctcc tcgttcgacc ccgcctcgat cctcccttta tccagccctc actccttctc
     1441 taggcgccgg aattagatct gcggccgcgc caccatggag aaggataaca ccgtgccact
     1501 gaaattgatt gccctgttag cgaacggtga atttcactct ggcgagcagt tgggtgaaac
     1561 gctgggaatg agccgggcgg ctattaataa acacattcag acactgcgtg actggggcgt
     1621 tgatgtcttt accgttccgg gtaaaggata cagcctgcct gagcctatcc agttacttaa
     1681 tgctaaacag atattgggtc agctggatgg cggtagtgta gccgtgctgc cagtgattga
     1741 ctccacgaat cagtaccttc ttgatcgtat cggagagctt aaatcgggcg atgcttgcat
     1801 tgcagaatac cagcaggctg gccgtggtcg ccggggtcgg aaatggtttt cgccttttgg
     1861 cgcaaactta tatttgtcga tgttctggcg tctggaacaa ggcccggcgg cggcgattgg
     1921 tttaagtctg gttatcggta tcgtgatggc ggaagtatta cgcaagctgg gtgcagataa
     1981 agttcgtgtt aaatggccta atgacctcta tctgcaggat cgcaagctgg caggcattct
     2041 ggtggagctg actggcaaaa ctggcgatgc ggcgcaaata gtcattggag ccgggatcaa
     2101 catggcaatg cgccgtgttg aagagagtgt cgttaatcag gggtggatca cgctgcagga
     2161 agcggggatc aatctcgatc gtaatacgtt ggcggccatg ctaatacgtg aattacgtgc
     2221 tgcgttggaa ctcttcgaac aagaaggatt ggcaccttat ctgtcgcgct gggaaaagct
     2281 ggataatttt attaatcgcc cagtgaaact tatcattggt gataaagaaa tatttggcat
     2341 ttcacgcgga atagacaaac agggggcttt attacttgag caggatggaa taataaaacc
     2401 ctggatgggc ggtgaaatat ccctgcgtag tgcagaaaaa ggcagcggcg ccaccaactt
     2461 cagcctgctg aagcaggccg gcgacgtgga ggagaacccc ggccctatgg tgagcaaggg
     2521 cgaggaggat aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg
     2581 ctccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg
     2641 cacccagacc gccaagctga aggtgaccaa gggtggcccc ctgcccttcg cctgggacat
     2701 cctgtcccct cagttcatgt acggctccaa ggcctacgtg aagcaccccg ccgacatccc
     2761 cgactacttg aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga
     2821 ggacggcggc gtggtgaccg tgacccagga ctcctccctg caggacggcg agttcatcta
     2881 caaggtgaag ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc agaagaagac
     2941 catgggctgg gaggcctcct ccgagcggat gtaccccgag gacggcgccc tgaagggcga
     3001 gatcaagcag aggctgaagc tgaaggacgg cggccactac gacgctgagg tcaagaccac
     3061 ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga
     3121 catcacctcc cacaacgagg actacaccat cgtggaacag tacgaacgcg ccgagggccg
     3181 ccactccacc ggcggcatgg acgagctgta caagtaagga tccgtcgacc tgcagccaag
     3241 cttatcgata aaataaaaga ttttatttag tctccagaaa aaggggggaa tgaaagaccc
     3301 cacctgtagg tttggcaagc tagcttaatt aacgccattt tgcaaggcat ggaaaataca
     3361 taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca gaatatgggc
     3421 caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga acagatggtc
     3481 cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt ccagggtgcc
     3541 ccaaggacct gaaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc
     3601 ttctgttcgc gcgcttctgc tccccgagct caataaaaga gcccacaacc cctcactcgg
     3661 cgcgccagtc ctccgataga ctgcgtcgcc cgggtacccg tgtatccaat aaaccctctt
     3721 gcagttgcat ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga
     3781 ctacccgtca gcgggggtct ttcatgggta acagtttctt gaagttggag aacaacattc
     3841 tgagggtagg agtcgaatat taagtaatcc tgactcaatt agccactgtt ttgaatccac
     3901 atactccaat actcctgaaa tagttcatta tggacagcgc agaagagctg gggagaatta
     3961 attcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     4021 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     4081 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4141 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     4201 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     4261 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     4321 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     4381 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     4441 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     4501 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     4561 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     4621 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     4681 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     4741 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     4801 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     4861 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     4921 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     4981 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5041 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     5101 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     5161 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     5221 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     5281 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     5341 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     5401 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     5461 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     5521 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     5581 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     5641 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     5701 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     5761 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     5821 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     5881 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     5941 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6001 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6061 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     6121 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     6181 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     6241 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     6301 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga
     6361 ttgtactgag agtgcaccat atgctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     6421 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     6481 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     6541 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     6601 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     6661 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     6721 aaaagctccc gggagcttgt atatccattt tcggatctga tcagcacgtg ttgacaatta
     6781 atcatcggca tagtatatcg gcatagtata atacgacaag gtgaggaacc atatgcggtg
     6841 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgccatt cgccattcag
     6901 gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc
     6961 gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg
     7021 acgttgtaaa acgacggcca gtgccaagct cgctctccct tatgcgactc ctgcattagg
     7081 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     7141 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     7201 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     7261 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     7321 aggatcgatt agtccaattt gttaaagaca ggatatcagt ggtccaggct ctagttttga
     7381 ctcaacaata tcaccagctg aagcctatag agtacgagcc ata
//
