LOCUS       sequence-217996-        6071 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 6071)
  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 6071)
  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--Grb2-4
FEATURES             Location/Qualifiers
     source          1..6071
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        380..583
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     534..554
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     628..647
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        628..646
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(700..702,703..705,706..1416)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(700..702,703..705,706..1416)
                     /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(745..766)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1006..1025)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1353..1374
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1682..1699)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1688..1912
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1958..2386
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2045..2064)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2255..2276
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2395..2415)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2400..2729
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2580..2715
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2642..2661
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2750..3349
                     /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(2750..2769)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3246..3266
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3509..3630
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3546..3565)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3600..3619
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3679..3695)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3679..3695)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3692..3714)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3703..3719
                     /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(3727..3733,3734..3751,3752..3757))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3772..3793
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3910..3927)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4081..4669)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4161..4180)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4840..5631,5632..5700))
                     /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     5463..5482
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5701..5805)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5880..5899)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(6071,1..379)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
       61 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
      121 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      181 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      241 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      301 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      361 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
      421 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
      481 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
      541 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga
      601 acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga cccaagctgg
      661 ctagcgttta aacttaagct tggtaccgag ctcggatcca tggtgagcaa gggcgaggag
      721 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
      781 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
      841 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
      901 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
      961 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     1021 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     1081 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     1141 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     1201 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     1261 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     1321 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     1381 gccgggatca ctctcggcat ggacgagctg tacaagggcc ggcctgtggt ggaattcatg
     1441 gaagccatcg ccaaatatga cttcaaagct actgctgacg atgagctgag cttcaaaagg
     1501 ggggacatcc ttaaggtttt gaatgaagaa tgtgaccaga actggtataa ggcagaactc
     1561 aatgggaaag atggcttcat ccccaagaat tacatagaaa tgaaaccaca tccgctcgag
     1621 gatacatcca gcacagtggc gcgcctcgag tctagagggc ccgtttaaac ccgctgatca
     1681 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     1741 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     1801 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     1861 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
     1921 gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta
     1981 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     2041 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     2101 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     2161 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     2221 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     2281 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     2341 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg
     2401 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     2461 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     2521 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     2581 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     2641 ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag
     2701 gcttttttgg aggcctaggc ttttgcaaaa agctcccggg gccgccacca tgaccgagta
     2761 caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc cgggccgtac gcaccctcgc
     2821 cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gacccggacc gccacatcga
     2881 gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt
     2941 gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc
     3001 gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc
     3061 cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt
     3121 cctggccacc gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt
     3181 gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc
     3241 gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt
     3301 gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgat tcgaaatgac
     3361 cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga
     3421 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     3481 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     3541 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     3601 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     3661 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     3721 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     3781 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     3841 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     3901 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     3961 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     4021 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     4081 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     4141 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     4201 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     4261 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     4321 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     4381 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     4441 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     4501 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     4561 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     4621 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     4681 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     4741 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     4801 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     4861 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     4921 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     4981 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     5041 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     5101 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     5161 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     5221 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     5281 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     5341 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     5401 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     5461 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     5521 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     5581 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     5641 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     5701 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     5761 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     5821 agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt
     5881 acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag
     5941 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat
     6001 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga
     6061 tatacgcgtt g
//
