LOCUS       sequence-265998-        5423 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Control plasmid for immunoprecipitation of DDK/FLAG-tagged proteins.
            Also a good control for transfection efficiency..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5423)
  AUTHORS   Stefely JA, Zhang Y, Freiberger EC, Kwiecien NW, Thomas HE, Davis 
            AM, Lowry ND, Vincent CE, Shishkova E, Clark NA, Medvedovic M, Coon 
            JJ, Pagliarini DJ, Mercer CA
  TITLE     Mass spectrometry proteomics reveals a function for mammalian 
            CALCOCO1 in MTOR-regulated selective autophagy.
  JOURNAL   Autophagy. 2020 Jan 23. doi: 10.1080/15548627.2020.1719746.
  PUBMED    31971854
REFERENCE   2  (bases 1 to 5423)
  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: "Autophagy. 
            2020 Jan 23. doi: 10.1080/15548627.2020.1719746."
COMMENT     Sequence Label: GFP-DDK
FEATURES             Location/Qualifiers
     source          1..5423
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        14..393
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        394..597
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     548..568
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     594..618
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        828..846
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     828..845
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     859..878
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             join(918..920,921..923,924..1634)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(918..920,921..923,924..1634)
                     /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(963..984)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1224..1243)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1571..1592
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1647..1670
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1683..1712
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     regulatory      1723..1732
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1736..1870
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1773..1792)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1827..1846
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        1939..2296
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1939..2130
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1962..1982)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2147..2282
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2316..2333)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2316..2332)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2325..2347)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2545..3000
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2632..2651)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2842..2863
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3017..3036)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3136..3158
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3196..3214)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3282..3386
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3387..3455,3456..4247)
                     /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     complement(3605..3624)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4418..5006
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4907..4926
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5160..5177
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5294..5315
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5330..5335,5336..5353,5354..5360)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5368..5384
                     /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)."
     primer_bind     5373..5395
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5392..5408
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5392..5408
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca
       61 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      121 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      181 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      241 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      301 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      361 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      421 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      481 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      541 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
      601 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
      661 caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt
      721 gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttcgtta
      781 gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac
      841 tatagaataa catccacttt gcctttctct ccacaggtgt ccacttccca ggtccaactg
      901 tacctcgcga gccagccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
      961 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     1021 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     1081 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     1141 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     1201 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     1261 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     1321 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     1381 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     1441 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     1501 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
     1561 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     1621 acgagctgta caagggcggc cgcatggact acaaggacga cgatgacaag tcatctagaa
     1681 tggagcagaa gctgatctcc gaggaggacc tgtgaaagct tggccgccat ggcccaactt
     1741 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     1801 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     1861 tgtctggatc gggaattaat tcggcgcagc accatggcct gaaataacct ctgaaagagg
     1921 aacttggtta ggtaccttct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg
     1981 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     2041 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     2101 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     2161 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     2221 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     2281 cctaggcttt tgcaaaaagc tgttaacagc ttggcactgg ccgtcgtttt acaacgtcgt
     2341 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     2401 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     2461 aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     2521 cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     2581 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     2641 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     2701 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     2761 tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     2821 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     2881 tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     2941 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
     3001 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
     3061 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     3121 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     3181 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     3241 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     3301 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     3361 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     3421 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     3481 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     3541 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     3601 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     3661 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     3721 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     3781 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     3841 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     3901 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     3961 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     4021 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     4081 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     4141 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     4201 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     4261 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     4321 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     4381 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     4441 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4501 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4561 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4621 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4681 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4741 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4801 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4861 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4921 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4981 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     5041 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     5101 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     5161 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
     5221 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     5281 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
     5341 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
     5401 gctatgacat gattacgaat taa
//
