LOCUS       sequence-308120-        6784 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express in mammalian cells TMEM97 and mCardianl to follow 
            transfection efficiency using flow cytometry. mCardinal is expressed
            following a P2A skip peptide so it is not connected to TMEM97..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6784)
  AUTHORS   Alon A, Schmidt HR, Wood MD, Sahn JJ, Martin SF, Kruse AC
  TITLE     Identification of the gene that codes for the sigma2 receptor.
  JOURNAL   Proc Natl Acad Sci U S A. 2017 Jul 3;114(27):7160-7165. doi: 
            10.1073/pnas.1705154114. Epub 2017 May 30.
  PUBMED    28559337
REFERENCE   2  (bases 1 to 6784)
  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"; doi: 
            "10.1073/pnas.1705154114"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2017-07-3- 3"; volume: "114"; issue: "27"; pages: "7160-7165"
COMMENT     Sequence Label: pTarget TMEM97-P2A-mCardinal
FEATURES             Location/Qualifiers
     source          1..6784
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..729
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     680..700
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     726..750
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          890..1022
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1005..1024
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     1191..1213
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1229..1248
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1229..1247
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1814..1870
                     /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             1871..2605
                     /codon_start=1
                     /product="far-red fluorescent protein derived from 
                     mNeptune, with further red-shifted spectra"
                     /label=mCardinal
                     /translation="MVSKGEELIKENMPMKLYMEGTVNNHHFKCTTEGEGKPYEGTQTQ
                     RIKVVEGGPLPFAFDILATCFMYGSKTFIKHPKGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTVTQDTSLQDGCLIYNVKLRGVNFPSNGPVMQKKTLGWEATTETLYPADGGLEGRCD
                     MALKLDGGGHLHCNLKTTYRSKKPAGNLKMPGVYFVDRRLERIKEADNETYVEQHEVAE
                     ARYCDLPSKLGHKLNGMDELYK"
     primer_bind     complement(2694..2716)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2705..2721
                     /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(2729..2735,2736..2753,2754..2759))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2774..2795
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     polyA_signal    2851..2972
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2862..2881)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2916..2935
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3106..3561
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3193..3212)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3403..3424
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3576..3933
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3576..3767
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3599..3619)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3784..3919
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3984..4583
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3984..4003)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4480..4500
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4647..4695
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(4722..4741)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4841..4863
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4901..4919)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4987..5091
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5092..5160,5161..5952)
                     /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     complement(5310..5329)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6123..6711
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6612..6631
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactg
      661 cgatcgcccg ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
      721 agcagagctc gtttagtgaa ccgtcagatc actagaagct ttattgcggt agtttatcac
      781 agttaaattg ctaacgcagt cagtgcttct gacacaacag tctcgaactt aagctgcagt
      841 gactctctta aggtagcctt gcagaagttg gtcgtgaggc actgggcagg taagtatcaa
      901 ggttacaaga caggtttaag gagaccaata gaaactgggc ttgtcgagac agagaagact
      961 cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct ttctctccac
     1021 aggtgtccac tcccagttca attacagctc ttaaaaattg gatctccatt cgccattcag
     1081 gctgcgcaac tgctgggaag gacgatcaga gcgggcctct tcgctattac gccagctggc
     1141 gaaagggacg tggcaagcaa ggcgattaag ttgagttacg ccaggatttt cccagtcacg
     1201 acgttgtaaa acgacggcca gagaattata atacgactca ctatagggcg aattcggatc
     1261 ccgacagact atgggggctc cggcaaccag gcgctgcgtg gagtggctgc tgggcctcta
     1321 cttcctcagc cacatcccca tcaccctgtt catggacctg caggcggtgc tgccgcgcga
     1381 gctctaccca gtcgagttta gaaacctgct gaagtggtat gctaaggagt tcaaagaccc
     1441 actgctacag gagcccccag cctggtttaa gtcctttctg ttttgcgagc ttgtgtttca
     1501 gctgcctttc tttcccattg caacgtatgc cttcctcaaa ggaagctgca agtggattcg
     1561 aactcctgca atcatctact ctgttcacac catgacaacc ttaattccga tactctccac
     1621 atttctgttt gaggatttct ccaaagccag tggtttcaag ggacaaagac ctgagacttt
     1681 gcatgaacgg ttaacccttg tgtctgtcta tgccccctac ttactcatcc cattcatact
     1741 tttaattttc atgttgcgga gcccctacta caagtatgaa gagaaaagaa agaagaaagg
     1801 taccggaagc ggagctacta acttcagcct gctgaagcag gctggagacg tggaggagaa
     1861 ccctggacct atggtgagca agggcgagga gctgatcaag gagaacatgc ccatgaagct
     1921 gtacatggaa ggcaccgtga acaaccacca cttcaagtgc accaccgaag gggagggcaa
     1981 gccctacgag ggcacccaga cccagaggat taaggtggtg gagggaggcc ccctgccgtt
     2041 cgcattcgac atcctggcca cctgctttat gtacgggagc aagaccttca tcaagcaccc
     2101 caagggcatc cccgatttct ttaagcagtc cttccctgag ggcttcacat gggagagagt
     2161 caccacatac gaagacgggg gcgtgcttac cgttacccag gacaccagcc tccaggacgg
     2221 ctgcttgatc tacaacgtca agctcagagg ggtgaacttc ccatccaacg gccctgtgat
     2281 gcagaagaaa acactcggct gggaggccac caccgagacc ctgtaccccg ctgacggcgg
     2341 cctggaaggc agatgcgaca tggccctgaa gctcgacggc gggggccacc tgcactgcaa
     2401 cctgaagacc acatacagat ccaagaaacc cgctggcaac ctcaagatgc ccggcgtcta
     2461 ctttgtggac cgcagactgg aaagaatcaa ggaggccgac aatgagacct acgtcgagca
     2521 gcacgaggtg gccgaggcca gatactgcga cctccctagc aaactggggc acaaacttaa
     2581 tggcatggac gagctgtaca agtaagtacc gtcgactgcg gccgcgaatt ccaagcttga
     2641 gtattctatc gtgtcaccta aataacttgg cgtaatcatg gtcatatctg tttcctgtgt
     2701 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     2761 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgcgat gcttccattt
     2821 tgtgagggtt aatgcttcga gaagacatga taagatacat tgatgagttt ggacaaacca
     2881 caacaagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
     2941 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt
     3001 ttcaggttca gggggagatg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg
     3061 gtaaaatccg ataaggatcg attccggagc ctgaatggcg aatggacgcg ccctgtagcg
     3121 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     3181 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     3241 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     3301 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     3361 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     3421 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     3481 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     3541 aaatattaac gcttacaatt tcgcctgtgt accttctgag gcggaaagaa ccagctgtgg
     3601 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3661 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     3721 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     3781 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     3841 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     3901 ggaggctttt ttggaggcct aggcttttgc aaaaagcttg attcttctga cacaacagtc
     3961 tcgaacttaa ggctagagcc accatgaccg agtacaagcc cacggtgcgc ctcgccaccc
     4021 gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca
     4081 cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct
     4141 tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg
     4201 tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc
     4261 cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc
     4321 tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg
     4381 accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc
     4441 gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc
     4501 ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca
     4561 tgacccgcaa gcccggtgcc tgagcgggac tctggggttc gaaatgaccg accaagcgac
     4621 gcccaacctg ccatcacgat ggccgcaata aaatatcttt attttcatta catctgtgtg
     4681 ttggtttttt gtgtgaagat ccgcgtatgg tgcactctca gtacaatctg ctctgatgcc
     4741 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     4801 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     4861 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     4921 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     4981 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     5041 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     5101 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     5161 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     5221 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     5281 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     5341 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     5401 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     5461 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     5521 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     5581 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     5641 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     5701 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     5761 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     5821 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     5881 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     5941 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     6001 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     6061 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     6121 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     6181 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     6241 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     6301 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     6361 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     6421 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     6481 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     6541 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     6601 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     6661 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     6721 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat ggctcgacag
     6781 atct
//
