LOCUS       sequence-273221-        5419 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Control plasmid for immunoprecipitation of HA-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 5419)
  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 5419)
  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: HA-GFP
FEATURES             Location/Qualifiers
     source          1..5419
                     /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             920..946
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(956..958,959..1669)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(956..958,959..1669)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(998..1019)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1259..1278)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1606..1627
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1679..1708
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     regulatory      1719..1728
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1732..1866
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1769..1788)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1823..1842
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        1935..2292
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1935..2126
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1958..1978)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2143..2278
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2312..2329)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2312..2328)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2321..2343)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2541..2996
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2628..2647)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2838..2859
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3013..3032)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3132..3154
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3192..3210)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3278..3382
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3383..3451,3452..4243)
                     /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(3601..3620)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4414..5002
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4903..4922
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5156..5173
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5290..5311
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5326..5331,5332..5349,5350..5356)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5364..5380
                     /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     5369..5391
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5388..5404
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5388..5404
                     /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 ccactcccag gtccaactgt
      901 acctcgcgag ccagccatgt acccatacga cgtcccagac tacgctggcg gccgcgtgag
      961 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     1021 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     1081 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     1141 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
     1201 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     1261 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     1321 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     1381 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     1441 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     1501 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     1561 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     1621 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt ctagaatgga
     1681 gcagaagctg atctccgagg aggacctgtg aaagcttggc cgccatggcc caacttgttt
     1741 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     1801 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     1861 tggatcggga attaattcgg cgcagcacca tggcctgaaa taacctctga aagaggaact
     1921 tggttaggta ccttctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt
     1981 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     2041 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     2101 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     2161 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     2221 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     2281 ggcttttgca aaaagctgtt aacagcttgg cactggccgt cgttttacaa cgtcgtgact
     2341 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     2401 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     2461 gcgaatggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     2521 tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     2581 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     2641 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     2701 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg
     2761 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     2821 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     2881 gggctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     2941 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg
     3001 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     3061 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     3121 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     3181 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     3241 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     3301 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     3361 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     3421 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     3481 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     3541 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     3601 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     3661 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     3721 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     3781 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     3841 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     3901 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     3961 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     4021 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     4081 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     4141 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     4201 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     4261 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     4321 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     4381 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     4441 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     4501 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     4561 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     4621 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     4681 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     4741 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     4801 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     4861 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     4921 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     4981 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     5041 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     5101 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     5161 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     5221 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     5281 caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct
     5341 tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta
     5401 tgacatgatt acgaattaa
//
