LOCUS       sequence-220589-        6363 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 6363)
  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 6363)
  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--Grb2-2
FEATURES             Location/Qualifiers
     source          1..6363
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(701..703,704..706,707..1417)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(701..703,704..706,707..1417)
                     /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(746..767)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1007..1026)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1354..1375
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1975..1992)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1981..2205
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2251..2679
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2338..2357)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2548..2569
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2688..2708)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2693..3022
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2873..3008
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2935..2954
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3043..3642
                     /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(3043..3062)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3539..3559
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3802..3923
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3839..3858)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3893..3912
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3972..3988)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3972..3988)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3985..4007)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3996..4012
                     /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(4020..4026,4027..4044,4045..4050))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4065..4086
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4203..4220)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4374..4962)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4454..4473)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5133..5924,5925..5993))
                     /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     5756..5775
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5994..6098)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6173..6192)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagcgttt aaacttaagc ttggtaccga gctcggatcc atggtgagca agggcgagga
      721 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
      781 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
      841 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
      901 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
      961 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     1021 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     1081 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     1141 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     1201 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     1261 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
     1321 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     1381 cgccgggatc actctcggca tggacgagct gtacaagggc cggcctgtgg tggaattccc
     1441 gtggtttttt ggcaaaatcc ccagagccaa ggcagaagaa atgctcagca aacagcggca
     1501 tgacggggcc ttcctgatcc gagagagcga gagcgctcct ggggacttct ccctgtccgt
     1561 caagtttgga aatgatgtgc agcacttcaa ggtgctccgc gacggagccg ggaagtattt
     1621 cctgtgggtg gtgaagttta attctttgaa tgagctggta gattaccaca gatcaacatc
     1681 cgtgtccagg aaccagcaga tattcttacg ggacatagaa cagatgccac agcagccaac
     1741 ctacgtccag gcgctctttg actttgaccc ccaggaggat ggcgagctgg gctttcgcag
     1801 aggagacttc attcatgtca tggataactc agatcccaat tggtggaaag gggcctgcca
     1861 cgggcagacc ggcatgtttc cccgcaatta tgtcaccccc gtgaaccgga acgtcgatat
     1921 ccagcacagt ggcgcgcctc gagtctagag ggcccgttta aacccgctga tcagcctcga
     1981 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     2041 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     2101 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     2161 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
     2221 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
     2281 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     2341 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     2401 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     2461 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     2521 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     2581 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     2641 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca
     2701 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     2761 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     2821 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     2881 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     2941 tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt
     3001 tggaggccta ggcttttgca aaaagctccc ggggccgcca ccatgaccga gtacaagccc
     3061 acggtgcgcc tcgccacccg cgacgacgtc ccccgggccg tacgcaccct cgccgccgcg
     3121 ttcgccgact accccgccac gcgccacacc gtcgacccgg accgccacat cgagcgggtc
     3181 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc
     3241 gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg
     3301 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag
     3361 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc
     3421 accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc
     3481 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc
     3541 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa
     3601 ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gattcgaaat gaccgaccaa
     3661 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg
     3721 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     3781 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     3841 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     3901 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     3961 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     4021 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     4081 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4141 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     4201 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     4261 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     4321 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     4381 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     4441 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     4501 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     4561 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     4621 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     4681 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     4741 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     4801 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     4861 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     4921 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     4981 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5041 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     5101 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     5161 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     5221 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     5281 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     5341 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     5401 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     5461 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     5521 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     5581 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     5641 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     5701 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     5761 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     5821 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     5881 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     5941 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6001 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     6061 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     6121 cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct
     6181 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg
     6241 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga
     6301 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc
     6361 gtt
//
