LOCUS       sequence-443430-        7366 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable expression of Btk1(PHD)-GFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7366)
  AUTHORS   Shinoda S, Sakai Y, Matsui T, Uematsu M, Koyama-Honda I, Sakamaki 
            JI, Yamamoto H, Mizushima N
  TITLE     Syntaxin 17 recruitment to mature autophagosomes is temporally 
            regulated by PI4P accumulation.
  JOURNAL   Elife. 2024 Jun 4;12:RP92189. doi: 10.7554/eLife.92189.
  PUBMED    38831696
REFERENCE   2  (bases 1 to 7366)
  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: "Elife."; 
            date: "2024-06-4"; volume: "12:RP92189. doi"; pages: " 
            10.7554/eLife.92189"
COMMENT     Sequence Label: pMRXIP-Btk1(PHD)-GFP
FEATURES             Location/Qualifiers
     source          1..7366
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    667..1024
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1089..1505
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1435..1457
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1473..1489
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    1515..1889
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     1827..1843
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     CDS             join(2479..2481,2482..2484,2485..3195)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2479..2481,2482..2484,2485..3195)
                     /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(2524..2545)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2785..2804)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3132..3153
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3233..3785
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3377..3394)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3604..3623
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3797..4396
                     /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(3797..3816)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4293..4313
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4620..5089
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5219..5236)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5390..5978)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5470..5489)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6142..6933,6934..7002))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6765..6784
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7003..7107)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7175..7193
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        join(7295..7366,1..308)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
       61 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
      121 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      181 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
      241 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      301 ttaccatgct agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct
      361 gtgccttatt tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttctgctc
      421 cccgagctca ataaaagagc ccacaacccc tcactcggcg cgccagtcct ccgattgact
      481 gagtcgcccg ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt
      541 ctcgctgttc cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt
      601 catttggggg ctcgtccggg atcgggagac ccctgcccag ggaccaccga cccaccaccg
      661 ggaggtaagc tggccagcaa cttatctgtg tctgtccgat tgtctagtgt ctatgactga
      721 ttttatgcgc ctgcgtcggt actagttagc taactagctc tgtatctggc ggacccgtgg
      781 tggaactgac gagttcggaa cacccggccg caaccctggg agacgtccca gggacttcgg
      841 gggccgtttt tgtggcccga cctgagtcca aaaatcccga tcgttttgga ctctttggtg
      901 cacccccctt agaggaggga tatgtggttc tggtaggaga cgagaaccta aaacagttcc
      961 cgcctccgtc tgaatttttg ctttcggttt gggaccgaag ccgcgccgcg cgtcttgtct
     1021 gctgcagcat cgttctgtgt tgtctctgtc tgactgtgtt tctgtatttg tctgaaaata
     1081 tgggcccggg ccagactgtt accactccct taagtttgac cttaggtcac tggaaagatg
     1141 tcgagcggat cgctcacaac cagtcggtag atgtcaagaa gagacgttgg gttaccttct
     1201 gctctgcaga atggccaacc tttaacgtcg gatggccgcg agacggcacc tttaaccgag
     1261 acctcatcac ccaggttaag atcaaggtct tttcacctgg cccgcatgga cacccagacc
     1321 aggtccccta catcgtgacc tgggaagcct tggcttttga cccccctccc tgggtcaagc
     1381 cctttgtaca ccctaagcct ccgcctcctc ttcctccatc cgccccgtct ctcccccttg
     1441 aacctcctcg ttcgaccccg cctcgatcct ccctttatcc agccctcact ccttctctag
     1501 gcgcccccat atggccatat gagatcttat atggggcacc cccgcccctt gtaaacttcc
     1561 ctgaccctga catgacaaga gttactaaca gcccctctct ccaagctcac ttacaggctc
     1621 tctacttagt ccagcacgaa gtctggagac ctctggcggc agcctaccaa gaacaactgg
     1681 accgaccggt ggtacctcac ccttaccgag tcggcgacac agtgtgggtc cgccgacacc
     1741 agactaagaa cctagaacct cgctggaaag gaccttacac agtcctgctg accaccccca
     1801 ccgccctcaa agtagacggc atcgcagctt ggatacacgc cgcccacgtg aaggctgccg
     1861 accccggggg tggaccatcc tctagactgc cggatcatcg atgaattcat ggccgcagtg
     1921 attctggaga gcatctttct gaagcgatcc caacagaaaa agaaaacatc acctctaaac
     1981 ttcaagaagc gcctgtttct cttgaccgtg cacaaactct cctactatga gtatgacttt
     2041 gaacgtggga gaagaggcag taagaagggt tcaatagatg ttgagaagat cacttgtgtt
     2101 gaaacagtgg ttcctgaaaa aaatcctcct ccagaaagac agattccgag aagaggtgaa
     2161 gagtccagtg aaatggagca aatttcaatc attgaaaggt tcccttatcc cttccaggtt
     2221 gtatatgatg aagggcctct ctacgtcttc tccccaactg aagaactaag gaagcggtgg
     2281 attcaccagc tcaaaaacgt aatccggtac aacagtgatc tggttcagaa atatcaccct
     2341 tgcttctgga tcgatgggca gtatctctgc tgctctcaga cagccaaaaa tgctatgggc
     2401 tgccaaattt tggagaacag gaatggaagc ttaaaacctg aattcactag tgttaacgga
     2461 tccctcgagc ggccgcccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     2521 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     2581 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     2641 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
     2701 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     2761 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     2821 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     2881 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     2941 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     3001 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     3061 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
     3121 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     3181 gacgagctgt acaagtawkm wggccgctac gtaaatwcck cccccccccc taacgttact
     3241 ggccgaagcc gcttggaata aggccggtgt gcgtttgtct atatgttatt ttccaccata
     3301 ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt gacgagcatt
     3361 cctaggggtc tttcccctct cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa
     3421 gcagttcctc tggaagcttc ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag
     3481 cggaaccccc cacctggcga caggtgcctc tgcggccaaa agccacgtgt ataagataca
     3541 cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc
     3601 aaatggctct cctcaagcgt attcaacaag gggctgaagg atgcccagaa ggtaccccat
     3661 tgtatgggat ctgatctggg gcctcggtgc acatgcttta catgtgttta gtcgaggtta
     3721 aaaaaacgtc taggcccccc gaaccacggg gacgtggttt tcctttgaaa aacacgatga
     3781 taatatggcc acaaccatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga
     3841 cgtcccccgg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca
     3901 caccgtcgac ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac
     3961 gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt
     4021 ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat
     4081 ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc
     4141 gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca
     4201 gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg
     4261 ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg
     4321 cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg
     4381 caagcccggt gcctgacgcc cgccccacga cccgcagcgc ccgaccgaaa ggagcgcacg
     4441 accccatggc tcgacgataa aataaaagat tttatttagt ctccagaaaa aggggggaat
     4501 gaaagacccc acctgtaggt ttggcaagct agcttaagta acgccatttt gcaaggcatg
     4561 gaaaaataca taactgagaa tagagaagtt cagatcaagg tcaggaacag atggaacagc
     4621 tgaatatggg ccaaacagga tatctgtggt aagcagttcc tgccccggct cagggccaag
     4681 aacagatgga acagctgaat atgggccaaa caggatatct gtggtaagca gttcctgccc
     4741 cggctcaggg ccaagaacag atggtcccca gatgcggtcc agccctcagc agtttctaga
     4801 gaaccatcag atgtttccag ggtgccccaa ggacctgaaa tgaccctgtg ccttatttga
     4861 actaaccaat cagttcgctt ctcgcttctg ttcgcgcgct tctgctcccc gagctcaata
     4921 aaagagccca caacccctca ctcggggcgc cagtcctccg attgactgag tcgcccgggt
     4981 acccgtgtat ccaataaacc ctcttgcagt tgcatccgac ttgtggtctc gctgttcctt
     5041 gggagggtct cctctgagtg attgactacc cgtcagcggg ggtctttcac atgcagcatg
     5101 tatcaaaatt aatttggttt tttttcttaa gtatttacat taaatggcca tagttgcatt
     5161 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     5221 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     5281 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     5341 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     5401 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     5461 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     5521 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     5581 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     5641 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     5701 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     5761 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     5821 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     5881 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     5941 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     6001 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     6061 caaaaaggat cttcacctag atccttttgc ggccggccgc aaatcaatct aaagtatata
     6121 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     6181 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6241 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     6301 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6361 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6421 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6481 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6541 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6601 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6661 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6721 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca cgggataata ccgcgccaca
     6781 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6841 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6901 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6961 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     7021 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     7081 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     7141 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     7201 tcttcactcg aggagcttgg cccattgcat acgttgtatc catatcataa tatgtacatt
     7261 tatattggct catgtccaac attaccgcca tgttgacatt gattattgac tagttattaa
     7321 tagtaatcaa ttacggggtc attagttcat agcccatata tggagt
//
