LOCUS       sequence-492996-        7462 bp    DNA     circular SYN 18-NOV-2025
DEFINITION  Inducible mCer3-TGN38-FAP expression in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7462)
  AUTHORS   Moore WM, Brea RJ, Knittel C, Wrightsman E, Hui B, Lou J, Ancajas 
            CF, Best MD, Obara, CJ Devaraj NK, Budin I
  TITLE     Leaflet-specific phospholipid imaging using genetically encoded 
            proximity sensors
  JOURNAL   Nat Chem Biol (2025). https://doi.org/10.1038/s41589-025-02021-z.
  PUBMED    38746395
REFERENCE   2  (bases 1 to 7462)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 18, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat Chem 
            Biol (2025). https://doi.org/10.1038/s41589-025-02021-z."
COMMENT     Sequence Label: pSBtet-mCer3-TGN38-FAP
FEATURES             Location/Qualifiers
     source          1..7462
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      451..1039
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     940..959
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1193..1210
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    1536..1760
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1749..1766
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(1821..2420)
                     /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(1904..1924)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     2401..2420
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             complement(2421..2477)
                     /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             complement(join(2484..2489,2490..2525,2526..2528,2529..2564,
                     2565..2567,2568..2603,2604..2609,2610..3227))
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     polyA_signal    3950..4071
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3987..4006)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4041..4060
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(5204..5914,5915..5917,5918..5920))
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5246..5267)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     5595..5614
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5854..5875
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      5917..5926
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6062..6374)
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     primer_bind     complement(6065..6089)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    6134..6152
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6170..6188
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6205..6223
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6241..6259
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6277..6295
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6312..6330
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6348..6366
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        6777..6881
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6882..6950,6951..7462,1..280)
                     /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(7100..7119)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
       61 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
      121 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
      181 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
      241 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
      301 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
      361 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
      421 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
      481 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
      541 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
      601 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
      661 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
      721 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
      781 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
      841 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
      901 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
      961 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     1021 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     1081 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     1141 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     1201 agtgagcgag gaagcggatc cctatacagt tgaagtcgga agtttacata caccttagcc
     1261 aaatacattt aaactcactt tttcacaatt cctgacattt aatcctagta aaaattccct
     1321 gtcttaggtc agttaggatc accactttat tttaagaatg tgaaatatca gaataatagt
     1381 agagagaatg attcatttca gcttttattt ctttcatcac attcccagtg ggtcagaagt
     1441 ttacatacac tcgactcctc tgcagaatgc ggcgatgttt cggtaagggg tcccgagtag
     1501 ctagtaggcc cagctggttc tttccgcctc agaagccata gagcccaccg catccccagc
     1561 atgcctgcta ttgtcttccc aatcctcccc cttgctgtcc tgccccaccc caccccccag
     1621 aatagaatga cacctactca gacaatgcga tgcaatttcc tcattttatt aggaaaggac
     1681 agtgggagtg gcaccttcca gggtcaagga aggcacgggg gaggggcaaa caacagatgg
     1741 ctggcaacta gaaggcacag tcgaggctga tcagcgagct ctagagaatt gatccccaag
     1801 cttcacgaca ggccttcgaa tcaggcaccg ggcttgcggg tcatgcacca ggtgcgcggt
     1861 ccttcgggca cctcgacgtc ggcggtgacg gtgaagccga gccgctcgta gaaggggagg
     1921 ttgcggggcg cggaggtctc caggaaggcg ggcaccccgg cgcgctcggc cgcctccact
     1981 ccggggagca cgacggcgct gcccagaccc ttgccctggt ggtcgggcga gacgccgacg
     2041 gtggccagga accacgcggg ctccttgggc cggtgcggcg ccaggaggcc ttccatctgt
     2101 tgctgcgcgg ccagccggga accgctcaac tcggccatgc gcgggccgat ctcggcgaac
     2161 accgcccccg cttcgacgct ctccggcgtg gtccagaccg ccaccgcggc gccgtcgtcc
     2221 gcgacccaca ccttgccgat gtcgagcccg acgcgcgtga ggaagagttc ttgcagctcg
     2281 gtgacccgct cgatgtggcg gtccggatcg acggtgtggc gcgtggcggg gtagtcggcg
     2341 aacgcggcgg cgagggtgcg tacggccctg gggacgtcgt cgcgggtggc gaggcgcacc
     2401 gtgggcttgt actcggtcat gggaccagga ttctcctcga cgtcaccagc ctgcttcagc
     2461 aggctgaagt tagtagcgcc ggacccgggg agcatgtcaa ggtcaaaatc gtcaagagcg
     2521 tcagcaggca gcatatcaag gtcaaagtcg tcaagggcat cggctgggag catgtctaag
     2581 tcaaaatcgt caagggcgtc ggccggcccg ccgctttcgc actttagctg tttctccagg
     2641 ccacatatga ttagttccag gccgaaaagg aaggcaggtt cggctccctg ccggtcgaac
     2701 agctcaattg cttgtctcag aagtgggggc atagaatcgg tggtaggtgt ctctctttcc
     2761 tcttttgcta cttgatgctc ctgttcctcc aatacgcagc ccagtgtaaa gtggcccacg
     2821 gcggacagag cgtacagtgc gttctccagg gagaagcctt gctgacacag gaacgcgagc
     2881 tgattttcca gggtttcgta ctgtttctct gttgggcggg tgccgagatg cactttagcc
     2941 ccgtcgcgat gtgagaggag agcacagcgg aatgacttgg cgttgttccg cagaaagtct
     3001 tgccatgact cgccttccag ggggcagaag tgggtatgat gcctgtccag catctcgatt
     3061 ggcagggcat cgagcagggc ccgcttgttc ttcacgtgcc agtacagggt aggctgctca
     3121 actcccagct tttgagcgag tttccttgtc gtcaggcctt cgataccgac tccattgagt
     3181 aattccagag cgccgtttat gactttgctc ttgtccagtc tagacatggt ggcctcaggt
     3241 gcagaggttt ctacagggaa agagagaaag gctacagtta gtttgctgtg atgatgctaa
     3301 gtgccgaggt cagatgtcta tggattgtac acggaatccc aacgcaggaa ccaaaaaaca
     3361 ctcacccaaa aaaatggcgc taaggccgag agaaaggcga agttccgtct acggctatat
     3421 accttgaaac aggggcggca gaagtgacgc aacgcggttg cgcggacggc ttcctgtgtt
     3481 ttcattggtc gaagcgttct gcgcgtgcgc agagattccc cagggaaggc gggccctctt
     3541 ccgaatgtcc aatccggaca ttcttccggt tggacctccg cggtcagcgc tgtgcccggc
     3601 gagagatcac gtggggcgcg gaggcggtgc tgctggggca cggccgtcca gcctcggcgg
     3661 ccatattttt gaggggctgt tcatctcgtt cacacgctct gtccgccatg tttgtgagtg
     3721 gaagcgccat taccccttca agcgactgaa ggctgcaggg cctctggtgg cccgcatggg
     3781 gagaccagac ccgccaggcc cgcctttccg cactcagtcc gggcttactt tattttgtga
     3841 gacagggtct cgcctaggta ccacatttgt agaggtttta cttgctttaa aaaacctccc
     3901 acatctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat
     3961 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     4021 tttttcactg cattcttgtt gtggtttgtc caaactcatc aatgtatctt atcatgtcta
     4081 tcgatggaag cttggcctga catcaaagct ttaggttcaa acgttggtag tcactggcct
     4141 ttggcctccg agtgactttg gatcttttat gggccagacc ggccgcggag acagtgacca
     4201 gggtaccctg gccccagtgc tggaaatact catccgaagg tattggacca ctattatcat
     4261 agtaatgtgt gggtccctcc ctcgcagcgt aatacacggc cgtgtccgcg gcggttacag
     4321 aggtcaactt caagaagaac tggttcttcg acttgtctga tgatatggtg actcgactct
     4381 tgagggccgg gttgtagtac gttctcccac tataatacat actcccaatc cactcaggcc
     4441 ccttccctgg gggctggcga atccagcccc agtagtaatg actactgctg atggaggcac
     4501 cagagacagt gcaggtgagg gacagggtct ccgaaggctt caccagtcct gggcccgact
     4561 cctgcagctg cacctgagaa ccgccgcctc cggaaccacc accgccgctg ccaccgccac
     4621 cagagccgcc accaccggaa actgtaacta aagtgccctg gccccaatgt tgaaaatact
     4681 catcgctagg tattgggcct gaattatcat aataatgcgt tgggccctca cgggcagcat
     4741 aatagacagc tgtatcagca gcggtaacgc tagtgagttt taaaaagaat tggttttttg
     4801 acttatcaga ggatatggta acacgtgact tcaaggcagg attatagtaa gttctacccg
     4861 agtagtacat agaccctatc cactctgggc cctttccagg tggttgtcta atccaacccc
     4921 aataatagtg cgaggaggaa attgatgcgc cactgactgt gcaggttagg gaaagtgtct
     4981 cagatggttt aaccaaacca ggtccagatt cttgcaactg gacttgcagg ccagaaaggc
     5041 cgaaagaccc accgcctcca gattccaggg caaaagcaat aatctttcgt ttgttgtggt
     5101 aagcaatata gaggacagca acaagaacag cagcggtcac cagataagca aagaagtggt
     5161 cgactgcaga attcgaagct tgagctcgag atctgagtcc ggacttgtac agctcgtcca
     5221 tgccgagagt gatcccggcg gcggtcacga actccagcag gaccatgtga tcgcgcttct
     5281 cgttggggtc tttgctcagc ttggactggg tgctcaggta gtggttgtcg ggcagcagca
     5341 cggggccgtc gccgatgggg gtgttctgct ggtagtggtc ggcgagctgc acgctgccgt
     5401 cctcgatgtt gtggcggatc ttgaagttgg ccttgatgcc gttcttctgc ttgtcggcgg
     5461 tgatatagac gttgtcgctg atggcgttgt actccagctt gtgccccagg atgttgccgt
     5521 cctccttgaa gtcgatgccc ttcagctcga tgcggttcac cagggtgtcg ccctcgaact
     5581 tcacctcggc gcgggtcttg tagttgccgt cgtccttgaa gaagatggtg cgctcctgga
     5641 cgtagccttc gggcatggcg gacttgaaga agtcgtgctg cttcatgtgg tcggggtagc
     5701 gggcgaagca ctgcacgccc caggtcaggg tggtcacgag ggtgggccag ggcacgggca
     5761 gcttgccggt ggtgcagatg aacttcaggg tcagcttgcc gtaggtggca tcgccctcgc
     5821 cctcgccgga cacgctgaac ttgtggccgt ttacgtcgcc gtccagctcg accaggatgg
     5881 gcaccacccc ggtgaacagc tcctcgccct tgctcaccat ggtggcgacc ggtgcgcaat
     5941 tcaccccgat gaataaaaaa gctaagtaca aaaggcactt catggtggcc tcagaggcct
     6001 ttcgagggta ggaagtggta cggaaagtag gtataaggca aaagagttgg aattggctcc
     6061 aggcgatctg acggttcact aaacgagctc tgcttttata ggcgcccacc gtacacgcct
     6121 aatcgacata cgttctctat cactgatagg gagtaaactc gacatacgtt ctctatcact
     6181 gatagggata aactcgacat acgttctcta tcactgatag ggagtaaact cgacatacgt
     6241 tctctatcac tgatagggag taaactcgac atacgttctc tatcactgat agggagtaaa
     6301 ctcgacatcg ttctctatca ctgataggga gtaaactcga catacgttct ctatcactga
     6361 tagggagtaa actcgtctag atagcggacc ccttaccgaa acatcgccgc attctgcaga
     6421 ggagtcgaac ttagtgtatg taaacttctg acccactgga attgtgatac agtgaattat
     6481 aagtgaaata atctgtctgt aaacaattgt tggaaaaatg acttgtgtca tgcacaaagt
     6541 agatgtccta actgacttgc caaaactatt gtttgttaac aagaaatttg tggagtagtt
     6601 gaaaaacgag ttttaatgac tccaacttaa gtgtatgtaa acttccgact tcaactgtat
     6661 agggatccaa ttttaacaaa atattaacgc ttacaatttc ctgatgcggt attttctcct
     6721 tacgcatctg tgcggtattt cacaccgcat caggtggcac ttttcgggga aatgtgcgcg
     6781 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     6841 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     6901 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     6961 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     7021 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     7081 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     7141 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     7201 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     7261 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     7321 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     7381 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     7441 cgttgcgcaa actattaact gg
//
