LOCUS       sequence-389402-        7427 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transposon vector (Addgene #60510) for doxycycline-inducible 
            expression of human PAG1 fused to EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7427)
  TITLE     Transposon vectors for proximity biotin labeling with split TurboID
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7427)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pSBtet-Bla/PAG1-GFP
FEATURES             Location/Qualifiers
     source          1..7427
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        295..607
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    303..321
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    339..357
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    374..392
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    410..428
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    446..464
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    481..499
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    517..535
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     580..604
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      680..689
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2012..2014,2015..2728)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIKV
                     NFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2054..2075)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2315..2334)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2662..2683
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2764..2885
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2775..2794)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2829..2848
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             join(3608..4225,4226..4231,4232..4267,4268..4270,4271..4306,
                     4307..4309,4310..4345,4346..4351)
                     /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"
     CDS             4358..4414
                     /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             4415..4813
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAMPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(4868..4885)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4874..5098
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5424..5441)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5595..6183)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5675..5694)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6354..7145,7146..7214))
                     /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     6977..6996
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7215..7319)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggatccctat acagttgaag tcggaagttt acatacactt aagttggagt cattaaaact
       61 cgtttttcaa ctactccaca aatttcttgt taacaaacaa tagttttggc aagtcagtta
      121 ggacatctac tttgtgcatg acacaagtca tttttccaac aattgtttac agacagatta
      181 tttcacttat aattcactgt atcacaattc cagtgggtca gaagtttaca tacactaagt
      241 tcgactcctc tgcagaatgc ggcgatgttt cggtaagggg tccgctatct agacgagttt
      301 actccctatc agtgatagag aacgtatgtc gagtttactc cctatcagtg atagagaacg
      361 atgtcgagtt tactccctat cagtgataga gaacgtatgt cgagtttact ccctatcagt
      421 gatagagaac gtatgtcgag tttactccct atcagtgata gagaacgtat gtcgagttta
      481 tccctatcag tgatagagaa cgtatgtcga gtttactccc tatcagtgat agagaacgta
      541 tgtcgattag gcgtgtacgg tgggcgccta taaaagcaga gctcgtttag tgaaccgtca
      601 gatcgcctgg agccaattcc aactcttttg ccttatacct actttccgta ccacttccta
      661 ccctcgaaag gcctctgagg ccaccatggg gcccgcgggg agcctgctgg gcagcggaca
      721 gatgcagatc accctgtggg gaagtctggc tgctgtcgcc attttcttcg tcatcacctt
      781 cctcatcttc ctgtgctcta gttgtgacag ggaaaagaag ccgcgacagc atagtgggga
      841 ccatgagaac ctgatgaacg tgccttcaga caaggagatg ttcagccgtt cagttactag
      901 cctggcaaca gatgctcctg ccagcagtga gcagaatggg gcactcacca atggggacat
      961 tctttcagag gacagtactc tgacctgcat gcagcattac gaggaagtcc agacatcggc
     1021 ctcggatctg ctggattccc aggacagcac agggaaacca aaatgtcatc agagtcggga
     1081 gctgcccaga atccctcccg agagcgcagt ggataccatg ctcacggcga gaagtgtgga
     1141 cggggaccag gggctgggga tggaagggcc ctatgaagtg ctcaaggaca gctcctccca
     1201 agaaaacatg gtggaggact gcttgtatga aactgtgaaa gagatcaagg aggtggctgc
     1261 agctgcacac ctggagaaag gccacagtgg caaggcaaaa tctacttctg cctcgaaaga
     1321 gctcccaggg ccccagactg aaggcaaagc tgagtttgct gaatatgcct cggtggacag
     1381 aaacaaaaaa tgtcgtcaaa gtgttaatgt agagagtatc cttggaaatt catgtgatcc
     1441 agaagaggag gccccaccac ctgtccctgt taagcttctg gacgagaatg aaaaccttca
     1501 ggagaaggaa gggggagagg cggaagagag tgccacagac acgaccagtg aaactaacaa
     1561 gagatttagc tcattgtcat acaagtctcg ggaagaagac cccactctca cagaagaaga
     1621 gatctcagct atgtactcat cagtaaataa acctggacag ttagtgaata aatcggggca
     1681 gtcgcttaca gttccggagt ccacctacac ctccattcaa ggggacccac agaggtcacc
     1741 ctcctcctgt aatgatctct atgctactgt taaagacttc gaaaaaactc caaacagcac
     1801 acttccacca gcagggaggc ccagcgagga gccagagcct gattatgaag cgatacagac
     1861 tctcaacaga gaggaagaaa aggccaccct ggggaccaat ggccaccacg gtctcgtccc
     1921 aaaggagaac gactacgaga gcataagtga cttgcagcaa ggcagagata ttaccaggct
     1981 cggaggaggc agcggtggtg gaagtggacc cgtgagcaag ggcgaggagc tgttcaccgg
     2041 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     2101 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
     2161 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ttcacctacg gcgtgcagtg
     2221 cttcgcccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     2281 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     2341 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     2401 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaaggt
     2461 ctatatcacc gccgacaagc agaagaacgg catcaaggtg aacttcaaga cccgccacaa
     2521 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     2581 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
     2641 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     2701 tctcggcatg gacgagctgt acaagtaagg cctgtcaggc caagcttcca tcgatagaca
     2761 tgataagata cattgatgag tttggacaaa ccacaacaag aatgcagtga aaaaaatgct
     2821 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
     2881 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag atgtgggagg
     2941 ttttttaaag caagtaaaac ctctacaaat gtggtaccta ggcgagaccc tgtctcacaa
     3001 aataaagtaa gcccggactg agtgcggaaa ggcgggcctg gcgggtctgg tctccccatg
     3061 cgggccacca gaggccctgc agccttcagt cgcttgaagg ggtaatggcg cttccactca
     3121 caaacatggc ggacagagcg tgtgaacgag atgaacagcc cctcaaaaat atggccgccg
     3181 aggctggacg gccgtgcccc agcagcaccg cctccgcgcc ccacgtgatc tctcgccggg
     3241 cacagcgctg accgcggagg tccaaccgga agaatgtccg gattggacat tcggaagagg
     3301 gcccgccttc cctggggaat ctctgcgcac gcgcagaacg cttcgaccaa tgaaaacaca
     3361 ggaagccgtc cgcgcaaccg cgttgcgtca cttctgccgc ccctgtttca aggtatatag
     3421 ccgtagacgg aacttcgcct ttctctcggc cttagcgcca tttttttggg tgagtgtttt
     3481 ttggttcctg cgttgggatt ccgtgtacaa tccatagaca tctgacctcg gcacttagca
     3541 tcatcacagc aaactaactg tagcctttct ctctttccct gtagaaacct ctgcacctga
     3601 ggccaccatg tctagactgg acaagagcaa agtcataaac ggcgctctgg aattactcaa
     3661 tggagtcggt atcgaaggcc tgacgacaag gaaactcgct caaaagctgg gagttgagca
     3721 gcctaccctg tactggcacg tgaagaacaa gcgggccctg ctcgatgccc tgccaatcga
     3781 gatgctggac aggcatcata cccacttctg ccccctggaa ggcgagtcat ggcaagactt
     3841 tctgcggaac aacgccaagt cattccgctg tgctctcctc tcacatcgcg acggggctaa
     3901 agtgcatctc ggcacccgcc caacagagaa acagtacgaa accctggaaa atcagctcgc
     3961 gttcctgtgt cagcaaggct tctccctgga gaacgcactg tacgctctgt ccgccgtggg
     4021 ccactttaca ctgggctgcg tattggagga acaggagcat caagtagcaa aagaggaaag
     4081 agagacacct accaccgatt ctatgccccc acttctgaga caagcaattg agctgttcga
     4141 ccggcaggga gccgaacctg ccttcctttt cggcctggaa ctaatcatat gtggcctgga
     4201 gaaacagcta aagtgcgaaa gcggcgggcc ggccgacgcc cttgacgatt ttgacttaga
     4261 catgctccca gccgatgccc ttgacgactt tgaccttgat atgctgcctg ctgacgctct
     4321 tgacgatttt gaccttgaca tgctccccgg gtccggcgct actaacttca gcctgctgaa
     4381 gcaggctggt gacgtcgagg agaatcctgg tcccatggcc atgcctttgt ctcaagaaga
     4441 atccaccctc attgaaagag caacggctac aatcaacagc atccccatct ctgaagacta
     4501 cagcgtcgcc agcgcagctc tctctagcga cggccgcatc ttcactggtg tcaatgtata
     4561 tcattttact gggggacctt gtgcagaact cgtggtgctg ggcactgctg ctgctgcggc
     4621 agctggcaac ctgacttgta tcgtcgcgat cggaaatgag aacaggggca tcttgagccc
     4681 ctgcggacgg tgccgacagg tgcttctcga tctgcatcct gggatcaaag ccatagtgaa
     4741 ggacagtgat ggacagccga cggcagttgg gattcgtgaa ttgctgccct ctggttatgt
     4801 gtgggagggc taattcgaag gcctgtcgtg aagcttgggg atcaattctc tagagctcgc
     4861 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     4921 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     4981 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     5041 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     5101 tctgaggcgg aaagaaccag ctgggcctac tagctactcg ggacccctta ccgaaacatc
     5161 gccgcattct gcagaggagt cgagtgtatg taaacttctg acccactggg aatgtgatga
     5221 aagaaataaa agctgaaatg aatcattctc tctactatta ttctgatatt tcacattctt
     5281 aaaataaagt ggtgatccta actgacctaa gacagggaat ttttactagg attaaatgtc
     5341 aggaattgtg aaaaagtgag tttaaatgta tttggctaag gtgtatgtaa acttccgact
     5401 tcaactgtat agggatccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     5461 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     5521 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     5581 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5641 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5701 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5761 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     5821 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5881 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     5941 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     6001 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     6061 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     6121 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     6181 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     6241 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     6301 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     6361 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     6421 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     6481 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     6541 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     6601 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     6661 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     6721 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     6781 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     6841 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     6901 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     6961 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     7021 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     7081 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     7141 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     7201 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     7261 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     7321 acatttcccc gaaaagtgcc acctgatgcg gtgtgaaata ccgcacagat gcgtaaggag
     7381 aaaataccgc atcaggaaat tgtaagcgtt aatattttgt taaaatt
//
