LOCUS       sequence-492998-        7429 bp    DNA     circular SYN 18-NOV-2025
DEFINITION  Inducible FAP-TGN38-GFP expression in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7429)
  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 7429)
  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-FAP-TGN38-GFP
FEATURES             Location/Qualifiers
     source          1..7429
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      307..895
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     796..815
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1049..1066
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    1392..1616
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1605..1622
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(1677..2276)
                     /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(1760..1780)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     2257..2276
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             complement(2277..2333)
                     /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(2340..2345,2346..2381,2382..2384,2385..2420,
                     2421..2423,2424..2459,2460..2465,2466..3083))
                     /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    3806..3927
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3843..3862)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3897..3916
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(3959..4672,4673..4675,4676..4678))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(3962..4672,4673..4675,4676..4678))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4004..4025)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     4353..4372
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4612..4633
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     promoter        complement(5885..6197)
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     primer_bind     complement(5888..5912)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    5957..5975
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5993..6011
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6028..6046
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6064..6082
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6100..6118
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6135..6153
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6171..6189
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        6600..6704
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6705..6773,6774..7429,1..136)
                     /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(6923..6942)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
       61 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
      121 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
      181 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
      241 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
      301 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
      361 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
      421 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
      481 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
      541 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
      601 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
      661 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
      721 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
      781 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
      841 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
      901 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
      961 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     1021 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggatcccta
     1081 tacagttgaa gtcggaagtt tacatacacc ttagccaaat acatttaaac tcactttttc
     1141 acaattcctg acatttaatc ctagtaaaaa ttccctgtct taggtcagtt aggatcacca
     1201 ctttatttta agaatgtgaa atatcagaat aatagtagag agaatgattc atttcagctt
     1261 ttatttcttt catcacattc ccagtgggtc agaagtttac atacactcga ctcctctgca
     1321 gaatgcggcg atgtttcggt aaggggtccc gagtagctag taggcccagc tggttctttc
     1381 cgcctcagaa gccatagagc ccaccgcatc cccagcatgc ctgctattgt cttcccaatc
     1441 ctcccccttg ctgtcctgcc ccaccccacc ccccagaata gaatgacacc tactcagaca
     1501 atgcgatgca atttcctcat tttattagga aaggacagtg ggagtggcac cttccagggt
     1561 caaggaaggc acgggggagg ggcaaacaac agatggctgg caactagaag gcacagtcga
     1621 ggctgatcag cgagctctag agaattgatc cccaagcttc acgacaggcc ttcgaatcag
     1681 gcaccgggct tgcgggtcat gcaccaggtg cgcggtcctt cgggcacctc gacgtcggcg
     1741 gtgacggtga agccgagccg ctcgtagaag gggaggttgc ggggcgcgga ggtctccagg
     1801 aaggcgggca ccccggcgcg ctcggccgcc tccactccgg ggagcacgac ggcgctgccc
     1861 agacccttgc cctggtggtc gggcgagacg ccgacggtgg ccaggaacca cgcgggctcc
     1921 ttgggccggt gcggcgccag gaggccttcc atctgttgct gcgcggccag ccgggaaccg
     1981 ctcaactcgg ccatgcgcgg gccgatctcg gcgaacaccg cccccgcttc gacgctctcc
     2041 ggcgtggtcc agaccgccac cgcggcgccg tcgtccgcga cccacacctt gccgatgtcg
     2101 agcccgacgc gcgtgaggaa gagttcttgc agctcggtga cccgctcgat gtggcggtcc
     2161 ggatcgacgg tgtggcgcgt ggcggggtag tcggcgaacg cggcggcgag ggtgcgtacg
     2221 gccctgggga cgtcgtcgcg ggtggcgagg cgcaccgtgg gcttgtactc ggtcatggga
     2281 ccaggattct cctcgacgtc accagcctgc ttcagcaggc tgaagttagt agcgccggac
     2341 ccggggagca tgtcaaggtc aaaatcgtca agagcgtcag caggcagcat atcaaggtca
     2401 aagtcgtcaa gggcatcggc tgggagcatg tctaagtcaa aatcgtcaag ggcgtcggcc
     2461 ggcccgccgc tttcgcactt tagctgtttc tccaggccac atatgattag ttccaggccg
     2521 aaaaggaagg caggttcggc tccctgccgg tcgaacagct caattgcttg tctcagaagt
     2581 gggggcatag aatcggtggt aggtgtctct ctttcctctt ttgctacttg atgctcctgt
     2641 tcctccaata cgcagcccag tgtaaagtgg cccacggcgg acagagcgta cagtgcgttc
     2701 tccagggaga agccttgctg acacaggaac gcgagctgat tttccagggt ttcgtactgt
     2761 ttctctgttg ggcgggtgcc gagatgcact ttagccccgt cgcgatgtga gaggagagca
     2821 cagcggaatg acttggcgtt gttccgcaga aagtcttgcc atgactcgcc ttccaggggg
     2881 cagaagtggg tatgatgcct gtccagcatc tcgattggca gggcatcgag cagggcccgc
     2941 ttgttcttca cgtgccagta cagggtaggc tgctcaactc ccagcttttg agcgagtttc
     3001 cttgtcgtca ggccttcgat accgactcca ttgagtaatt ccagagcgcc gtttatgact
     3061 ttgctcttgt ccagtctaga catggtggcc tcaggtgcag aggtttctac agggaaagag
     3121 agaaaggcta cagttagttt gctgtgatga tgctaagtgc cgaggtcaga tgtctatgga
     3181 ttgtacacgg aatcccaacg caggaaccaa aaaacactca cccaaaaaaa tggcgctaag
     3241 gccgagagaa aggcgaagtt ccgtctacgg ctatatacct tgaaacaggg gcggcagaag
     3301 tgacgcaacg cggttgcgcg gacggcttcc tgtgttttca ttggtcgaag cgttctgcgc
     3361 gtgcgcagag attccccagg gaaggcgggc cctcttccga atgtccaatc cggacattct
     3421 tccggttgga cctccgcggt cagcgctgtg cccggcgaga gatcacgtgg ggcgcggagg
     3481 cggtgctgct ggggcacggc cgtccagcct cggcggccat atttttgagg ggctgttcat
     3541 ctcgttcaca cgctctgtcc gccatgtttg tgagtggaag cgccattacc ccttcaagcg
     3601 actgaaggct gcagggcctc tggtggcccg catggggaga ccagacccgc caggcccgcc
     3661 tttccgcact cagtccgggc ttactttatt ttgtgagaca gggtctcgcc taggtaccac
     3721 atttgtagag gttttacttg ctttaaaaaa cctcccacat ctccccctga acctgaaaca
     3781 taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata
     3841 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt cttgttgtgg
     3901 tttgtccaaa ctcatcaatg tatcttatca tgtctatcga tggaagcttg gcctgacact
     3961 acttgtacag ctcgtccatg ccgagagtga tcccggcggc ggtcacgaac tccagcagga
     4021 ccatgtgatc gcgcttctcg ttggggtctt tgctcagggc ggactgggtg ctcaggtagt
     4081 ggttgtcggg cagcagcacg gggccgtcgc cgatgggggt gttctgctgg tagtggtcgg
     4141 cgagctgcac gctgccgtcc tcgatgttgt ggcggatctt gaagttcacc ttgatgccgt
     4201 tcttctgctt gtcggccatg atatagacgt tgtggctgtt gtagttgtac tccagcttgt
     4261 gccccaggat gttgccgtcc tccttgaagt cgatgccctt cagctcgatg cggttcacca
     4321 gggtgtcgcc ctcgaacttc acctcggcgc gggtcttgta gttgccgtcg tccttgaaga
     4381 agatggtgcg ctcctggacg tagccttcgg gcatggcgga cttgaagaag tcgtgctgct
     4441 tcatgtggtc ggggtagcgg ctgaagcact gcacgccgta ggtcagggtg gtcacgaggg
     4501 tgggccaggg cacgggcagc ttgccggtgg tgcagatgaa cttcagggtc agcttgccgt
     4561 aggtggcatc gccctcgccc tcgccggaca cgctgaactt gtggccgttt acgtcgccgt
     4621 ccagctcgac caggatgggc accaccccgg tgaacagctc ctcgcccttg ctcaccatgc
     4681 caccaccaag ctttaggttc aaacgttggt agtcactggc ctttggcctc cgagtgactt
     4741 tggatctttt tccttccagg gcaaaagcaa taatctttcg tttgttgtgg taagcaatat
     4801 agaggacagc aacaagaaca gcagcggtca ccagataagc aaagaagtga ccagatccag
     4861 acccatgggc cagaccggcc gcggagacag tgaccagggt accctggccc cagtgctgga
     4921 aatactcatc cgaaggtatt ggaccactat tatcatagta atgtgtgggt ccctccctcg
     4981 cagcgtaata cacggccgtg tccgcggcgg ttacagaggt caacttcaag aagaactggt
     5041 tcttcgactt gtctgatgat atggtgactc gactcttgag ggccgggttg tagtacgttc
     5101 tcccactata atacatactc ccaatccact caggcccctt ccctgggggc tggcgaatcc
     5161 agccccagta gtaatgacta ctgctgatgg aggcaccaga gacagtgcag gtgagggaca
     5221 gggtctccga aggcttcacc agtcctgggc ccgactcctg cagctgcacc tgagaaccgc
     5281 cgcctccgga accaccaccg ccgctgccac cgccaccaga gccgccacca ccggaaactg
     5341 taactaaagt gccctggccc caatgttgaa aatactcatc gctaggtatt gggcctgaat
     5401 tatcataata atgcgttggg ccctcacggg cagcataata gacagctgta tcagcagcgg
     5461 taacgctagt gagttttaaa aagaattggt tttttgactt atcagaggat atggtaacac
     5521 gtgacttcaa ggcaggatta tagtaagttc tacccgagta gtacatagac cctatccact
     5581 ctgggccctt tccaggtggt tgtctaatcc aaccccaata atagtgcgag gaggaaattg
     5641 atgcgccact gactgtgcag gttagggaaa gtgtctcaga tggtttaacc aaaccaggtc
     5701 cagattcttg caactggact tgcaggccag aaaggccgaa agaggtggcg accggtgcgc
     5761 aattcacccc gatgaataaa aaagctaagt acaaaaggca cttcatggtg gcctcagagg
     5821 cctttcgagg gtaggaagtg gtacggaaag taggtataag gcaaaagagt tggaattggc
     5881 tccaggcgat ctgacggttc actaaacgag ctctgctttt ataggcgccc accgtacacg
     5941 cctaatcgac atacgttctc tatcactgat agggagtaaa ctcgacatac gttctctatc
     6001 actgataggg ataaactcga catacgttct ctatcactga tagggagtaa actcgacata
     6061 cgttctctat cactgatagg gagtaaactc gacatacgtt ctctatcact gatagggagt
     6121 aaactcgaca tcgttctcta tcactgatag ggagtaaact cgacatacgt tctctatcac
     6181 tgatagggag taaactcgtc tagatagcgg accccttacc gaaacatcgc cgcattctgc
     6241 agaggagtcg aacttagtgt atgtaaactt ctgacccact ggaattgtga tacagtgaat
     6301 tataagtgaa ataatctgtc tgtaaacaat tgttggaaaa atgacttgtg tcatgcacaa
     6361 agtagatgtc ctaactgact tgccaaaact attgtttgtt aacaagaaat ttgtggagta
     6421 gttgaaaaac gagttttaat gactccaact taagtgtatg taaacttccg acttcaactg
     6481 tatagggatc caattttaac aaaatattaa cgcttacaat ttcctgatgc ggtattttct
     6541 ccttacgcat ctgtgcggta tttcacaccg catcaggtgg cacttttcgg ggaaatgtgc
     6601 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     6661 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     6721 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     6781 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     6841 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     6901 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     6961 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     7021 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     7081 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     7141 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     7201 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     7261 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     7321 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     7381 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcgg
//
