LOCUS       sequence-492997-        7483 bp    DNA     circular SYN 18-NOV-2025
DEFINITION  Inducible GFP-TGN38-FAP expression in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7483)
  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 7483)
  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-GFP-TGN38-FAP
FEATURES             Location/Qualifiers
     source          1..7483
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(11..323)
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     primer_bind     complement(14..38)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    83..101
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    119..137
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    154..172
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    190..208
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    226..244
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    261..279
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    297..315
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        726..830
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(831..899,900..1691)
                     /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(1049..1068)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1862..2450
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2351..2370
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2604..2621
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    2947..3171
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3160..3177
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(3232..3831)
                     /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(3315..3335)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     3812..3831
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             complement(3832..3888)
                     /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(3895..3900,3901..3936,3937..3939,3940..3975,
                     3976..3978,3979..4014,4015..4020,4021..4638))
                     /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    5361..5482
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5398..5417)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5452..5471
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(6636..7346,7347..7349,7350..7352))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(6636..7346,7347..7349,7350..7352))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6678..6699)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     7027..7046
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7286..7307
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      7349..7358
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 attggctcca ggcgatctga cggttcacta aacgagctct gcttttatag gcgcccaccg
       61 tacacgccta atcgacatac gttctctatc actgataggg agtaaactcg acatacgttc
      121 tctatcactg atagggataa actcgacata cgttctctat cactgatagg gagtaaactc
      181 gacatacgtt ctctatcact gatagggagt aaactcgaca tacgttctct atcactgata
      241 gggagtaaac tcgacatcgt tctctatcac tgatagggag taaactcgac atacgttctc
      301 tatcactgat agggagtaaa ctcgtctaga tagcggaccc cttaccgaaa catcgccgca
      361 ttctgcagag gagtcgaact tagtgtatgt aaacttctga cccactggaa ttgtgataca
      421 gtgaattata agtgaaataa tctgtctgta aacaattgtt ggaaaaatga cttgtgtcat
      481 gcacaaagta gatgtcctaa ctgacttgcc aaaactattg tttgttaaca agaaatttgt
      541 ggagtagttg aaaaacgagt tttaatgact ccaacttaag tgtatgtaaa cttccgactt
      601 caactgtata gggatccaat tttaacaaaa tattaacgct tacaatttcc tgatgcggta
      661 ttttctcctt acgcatctgt gcggtatttc acaccgcatc aggtggcact tttcggggaa
      721 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
      781 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
      841 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
      901 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
      961 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     1021 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     1081 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     1141 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     1201 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     1261 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     1321 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     1381 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     1441 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     1501 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     1561 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     1621 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     1681 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     1741 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     1801 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     1861 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     1921 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     1981 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     2041 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     2101 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     2161 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     2221 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     2281 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     2341 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     2401 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     2461 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     2521 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     2581 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggat ccctatacag
     2641 ttgaagtcgg aagtttacat acaccttagc caaatacatt taaactcact ttttcacaat
     2701 tcctgacatt taatcctagt aaaaattccc tgtcttaggt cagttaggat caccacttta
     2761 ttttaagaat gtgaaatatc agaataatag tagagagaat gattcatttc agcttttatt
     2821 tctttcatca cattcccagt gggtcagaag tttacataca ctcgactcct ctgcagaatg
     2881 cggcgatgtt tcggtaaggg gtcccgagta gctagtaggc ccagctggtt ctttccgcct
     2941 cagaagccat agagcccacc gcatccccag catgcctgct attgtcttcc caatcctccc
     3001 ccttgctgtc ctgccccacc ccacccccca gaatagaatg acacctactc agacaatgcg
     3061 atgcaatttc ctcattttat taggaaagga cagtgggagt ggcaccttcc agggtcaagg
     3121 aaggcacggg ggaggggcaa acaacagatg gctggcaact agaaggcaca gtcgaggctg
     3181 atcagcgagc tctagagaat tgatccccaa gcttcacgac aggccttcga atcaggcacc
     3241 gggcttgcgg gtcatgcacc aggtgcgcgg tccttcgggc acctcgacgt cggcggtgac
     3301 ggtgaagccg agccgctcgt agaaggggag gttgcggggc gcggaggtct ccaggaaggc
     3361 gggcaccccg gcgcgctcgg ccgcctccac tccggggagc acgacggcgc tgcccagacc
     3421 cttgccctgg tggtcgggcg agacgccgac ggtggccagg aaccacgcgg gctccttggg
     3481 ccggtgcggc gccaggaggc cttccatctg ttgctgcgcg gccagccggg aaccgctcaa
     3541 ctcggccatg cgcgggccga tctcggcgaa caccgccccc gcttcgacgc tctccggcgt
     3601 ggtccagacc gccaccgcgg cgccgtcgtc cgcgacccac accttgccga tgtcgagccc
     3661 gacgcgcgtg aggaagagtt cttgcagctc ggtgacccgc tcgatgtggc ggtccggatc
     3721 gacggtgtgg cgcgtggcgg ggtagtcggc gaacgcggcg gcgagggtgc gtacggccct
     3781 ggggacgtcg tcgcgggtgg cgaggcgcac cgtgggcttg tactcggtca tgggaccagg
     3841 attctcctcg acgtcaccag cctgcttcag caggctgaag ttagtagcgc cggacccggg
     3901 gagcatgtca aggtcaaaat cgtcaagagc gtcagcaggc agcatatcaa ggtcaaagtc
     3961 gtcaagggca tcggctggga gcatgtctaa gtcaaaatcg tcaagggcgt cggccggccc
     4021 gccgctttcg cactttagct gtttctccag gccacatatg attagttcca ggccgaaaag
     4081 gaaggcaggt tcggctccct gccggtcgaa cagctcaatt gcttgtctca gaagtggggg
     4141 catagaatcg gtggtaggtg tctctctttc ctcttttgct acttgatgct cctgttcctc
     4201 caatacgcag cccagtgtaa agtggcccac ggcggacaga gcgtacagtg cgttctccag
     4261 ggagaagcct tgctgacaca ggaacgcgag ctgattttcc agggtttcgt actgtttctc
     4321 tgttgggcgg gtgccgagat gcactttagc cccgtcgcga tgtgagagga gagcacagcg
     4381 gaatgacttg gcgttgttcc gcagaaagtc ttgccatgac tcgccttcca gggggcagaa
     4441 gtgggtatga tgcctgtcca gcatctcgat tggcagggca tcgagcaggg cccgcttgtt
     4501 cttcacgtgc cagtacaggg taggctgctc aactcccagc ttttgagcga gtttccttgt
     4561 cgtcaggcct tcgataccga ctccattgag taattccaga gcgccgttta tgactttgct
     4621 cttgtccagt ctagacatgg tggcctcagg tgcagaggtt tctacaggga aagagagaaa
     4681 ggctacagtt agtttgctgt gatgatgcta agtgccgagg tcagatgtct atggattgta
     4741 cacggaatcc caacgcagga accaaaaaac actcacccaa aaaaatggcg ctaaggccga
     4801 gagaaaggcg aagttccgtc tacggctata taccttgaaa caggggcggc agaagtgacg
     4861 caacgcggtt gcgcggacgg cttcctgtgt tttcattggt cgaagcgttc tgcgcgtgcg
     4921 cagagattcc ccagggaagg cgggccctct tccgaatgtc caatccggac attcttccgg
     4981 ttggacctcc gcggtcagcg ctgtgcccgg cgagagatca cgtggggcgc ggaggcggtg
     5041 ctgctggggc acggccgtcc agcctcggcg gccatatttt tgaggggctg ttcatctcgt
     5101 tcacacgctc tgtccgccat gtttgtgagt ggaagcgcca ttaccccttc aagcgactga
     5161 aggctgcagg gcctctggtg gcccgcatgg ggagaccaga cccgccaggc ccgcctttcc
     5221 gcactcagtc cgggcttact ttattttgtg agacagggtc tcgcctaggt accacatttg
     5281 tagaggtttt acttgcttta aaaaacctcc cacatctccc cctgaacctg aaacataaaa
     5341 tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca
     5401 atagcatcac aaatttcaca aataaagcat ttttttcact gcattcttgt tgtggtttgt
     5461 ccaaactcat caatgtatct tatcatgtct atcgatggaa gcttggcctg acattatcta
     5521 gatccggtgg atccatgggc cagaccggcc gcggagacag tgaccagggt accctggccc
     5581 cagtgctgga aatactcatc cgaaggtatt ggaccactat tatcatagta atgtgtgggt
     5641 ccctccctcg cagcgtaata cacggccgtg tccgcggcgg ttacagaggt caacttcaag
     5701 aagaactggt tcttcgactt gtctgatgat atggtgactc gactcttgag ggccgggttg
     5761 tagtacgttc tcccactata atacatactc ccaatccact caggcccctt ccctgggggc
     5821 tggcgaatcc agccccagta gtaatgacta ctgctgatgg aggcaccaga gacagtgcag
     5881 gtgagggaca gggtctccga aggcttcacc agtcctgggc ccgactcctg cagctgcacc
     5941 tgagaaccgc cgcctccgga accaccaccg ccgctgccac cgccaccaga gccgccacca
     6001 ccggaaactg taactaaagt gccctggccc caatgttgaa aatactcatc gctaggtatt
     6061 gggcctgaat tatcataata atgcgttggg ccctcacggg cagcataata gacagctgta
     6121 tcagcagcgg taacgctagt gagttttaaa aagaattggt tttttgactt atcagaggat
     6181 atggtaacac gtgacttcaa ggcaggatta tagtaagttc tacccgagta gtacatagac
     6241 cctatccact ctgggccctt tccaggtggt tgtctaatcc aaccccaata atagtgcgag
     6301 gaggaaattg atgcgccact gactgtgcag gttagggaaa gtgtctcaga tggtttaacc
     6361 aaaccaggtc cagattcttg caactggact tgcaggccag aaaggccgaa agacccaccg
     6421 cctccagaaa gctttaggtt caaacgttgg tagtcactgg cctttggcct ccgagtgact
     6481 ttggatcttt ttccttccag ggcaaaagca ataatctttc gtttgttgtg gtaagcaata
     6541 tagaggacag caacaagaac agcagcggtc accagataag caaagaagtg gtcgactgca
     6601 gaattcgaag cttgagctcg agatctgagt ccggacttgt acagctcgtc catgccgaga
     6661 gtgatcccgg cggcggtcac gaactccagc aggaccatgt gatcgcgctt ctcgttgggg
     6721 tctttgctca gggcggactg ggtgctcagg tagtggttgt cgggcagcag cacggggccg
     6781 tcgccgatgg gggtgttctg ctggtagtgg tcggcgagct gcacgctgcc gtcctcgatg
     6841 ttgtggcgga tcttgaagtt caccttgatg ccgttcttct gcttgtcggc catgatatag
     6901 acgttgtggc tgttgtagtt gtactccagc ttgtgcccca ggatgttgcc gtcctccttg
     6961 aagtcgatgc ccttcagctc gatgcggttc accagggtgt cgccctcgaa cttcacctcg
     7021 gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg tgcgctcctg gacgtagcct
     7081 tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt ggtcggggta gcggctgaag
     7141 cactgcacgc cgtaggtcag ggtggtcacg agggtgggcc agggcacggg cagcttgccg
     7201 gtggtgcaga tgaacttcag ggtcagcttg ccgtaggtgg catcgccctc gccctcgccg
     7261 gacacgctga acttgtggcc gtttacgtcg ccgtccagct cgaccaggat gggcaccacc
     7321 ccggtgaaca gctcctcgcc cttgctcacc atggtggcga ccggtgcgca attcaccccg
     7381 atgaataaaa aagctaagta caaaaggcac ttcatggtgg cctcagaggc ctttcgaggg
     7441 taggaagtgg tacggaaagt aggtataagg caaaagagtt gga
//
