LOCUS       sequence-217944-        6537 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  transient/stable overexpression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6537)
  AUTHORS   Gingras J, Gawor M, Bernadzki KM, Grady RM, Hallock P, Glass DJ, 
            Sanes JR, Proszynski TJ
  TITLE     Alpha-Dystrobrevin-1 recruits Grb2 and alpha-catulin to organize 
            neurotransmitter receptors at the neuromuscular junction.
  JOURNAL   J Cell Sci. 2016 Mar 1;129(5):898-911. doi: 10.1242/jcs.181180. Epub
            2016 Jan 14.
  PUBMED    26769899
REFERENCE   2  (bases 1 to 6537)
  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"; doi: 
            "10.1242/jcs.181180"; journalName: "J Cell Sci"; date: "2016-03-1- 
            1"; volume: "129"; issue: "5"; pages: "898-911"
COMMENT     Sequence Label: pcDNA3.1(+)/Puro/FA/GFP-N--Grb2-5
FEATURES             Location/Qualifiers
     source          1..6537
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        15..394
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        395..598
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     549..569
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     643..662
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        643..661
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(715..717,718..720,721..1431)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(715..717,718..720,721..1431)
                     /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(760..781)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1021..1040)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1368..1389
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2163..2180)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2169..2393
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2439..2867
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2526..2545)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2736..2757
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2876..2896)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2881..3210
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3061..3196
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3123..3142
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3231..3830
                     /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(3231..3250)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3727..3747
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3990..4111
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4027..4046)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4081..4100
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4160..4176)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4160..4176)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4173..4195)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4184..4200
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4208..4214,4215..4232,4233..4238))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4253..4274
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4391..4408)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4562..5150)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4642..4661)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5321..6112,6113..6181))
                     /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     5944..5963
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6182..6286)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6361..6380)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc
       61 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
      121 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
      181 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
      241 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
      301 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
      361 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
      421 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
      481 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
      541 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct
      601 ctggctaact agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag
      661 ggagacccaa gctggctagc gtttaaactt aagcttggta ccgagctcgg atccatggtg
      721 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
      781 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
      841 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
      901 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
      961 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     1021 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     1081 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     1141 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     1201 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     1261 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     1321 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     1381 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gggccggcct
     1441 gtggtggaat tcatggaagc catcgccaaa tatgacttca aagctactgc tgacgatgag
     1501 ctgagcttca aaagggggga catccttaag gttttgaatg aagaatgtga ccagaactgg
     1561 tataaggcag aactcaatgg gaaagatggc ttcatcccca agaattacat agaaatgaaa
     1621 ccacatccgt ggttttttgg caaaatcccc agagccaagg cagaagaaat gctcagcaaa
     1681 cagcggcatg acggggcctt cctgatccga gagagcgaga gcgctcctgg ggacttctcc
     1741 ctgtccgtca agtttggaaa tgatgtgcag cacttcaagg tgctccgcga cggagccggg
     1801 aagtatttcc tgtgggtggt gaagtttaat tctttgaatg agctggtaga ttaccacaga
     1861 tcaacatccg tgtccaggaa ccagcagata ttcttacggg acatagaaca gatgccacag
     1921 cagccaacct acgtccaggc gctctttgac tttgaccccc aggaggatgg cgagctgggc
     1981 tttcgcagag gagacttcat tcatgtcatg gataactcag atcccaattg gtggaaaggg
     2041 gcctgccacg ggcagaccgg catgtttccc cgcaattatg tcacccccgt gaaccggaac
     2101 gtcgatatcc agcacagtgg cgcgcctcga gtctagaggg cccgtttaaa cccgctgatc
     2161 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     2221 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     2281 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     2341 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     2401 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     2461 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     2521 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     2581 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     2641 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     2701 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     2761 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     2821 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     2881 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     2941 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     3001 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     3061 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     3121 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     3181 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg ggccgccacc atgaccgagt
     3241 acaagcccac ggtgcgcctc gccacccgcg acgacgtccc ccgggccgta cgcaccctcg
     3301 ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgacccggac cgccacatcg
     3361 agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg
     3421 tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag
     3481 cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg
     3541 ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt
     3601 tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg
     3661 tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg
     3721 cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg
     3781 tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga ttcgaaatga
     3841 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     3901 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     3961 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     4021 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     4081 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     4141 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     4201 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     4261 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4321 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     4381 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4441 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4501 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4561 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4621 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4681 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4741 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4801 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4861 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4921 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4981 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     5041 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     5101 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5161 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5221 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     5281 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     5341 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     5401 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     5461 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     5521 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5581 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     5641 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     5701 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     5761 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     5821 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     5881 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     5941 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     6001 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     6061 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     6121 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     6181 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     6241 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     6301 aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag
     6361 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga
     6421 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa
     6481 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggc
//
