LOCUS       sequence-335136-        7025 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of His-Cavin1-GFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7025)
  AUTHORS   Prasai B, Haber GJ, Strub MP, Ahn R, Ciemniecki JA, Sochacki KA, 
            Taraska JW
  TITLE     The nanoscale molecular morphology of docked exocytic dense-core 
            vesicles in neuroendocrine cells.
  JOURNAL   Nat Commun. 2021 Jun 25;12(1):3970. doi: 10.1038/s41467-021-24167-9.
  PUBMED    34172739
REFERENCE   2  (bases 1 to 7025)
  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: "Nat 
            Commun."; date: "2021-06-25"; volume: "12(1):3970. doi"; pages: " 
            10.1038/s41467-021-24167-9"
COMMENT     Sequence Label: His-Cavin1-GFP
FEATURES             Location/Qualifiers
     source          1..7025
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(12..31)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        203..582
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        583..786
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     737..757
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    788..806
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    809..827
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     837..861
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             959..976
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     regulatory      2174..2183
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2180..2182,2183..2185,2186..2899)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLK
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2225..2246)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2486..2505)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2945..2962)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2951..3175
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3308..3327)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3459..3506
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3514..4536
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3514..4536
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4666..4787
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4703..4722)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4757..4776
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4836..4852)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4836..4852)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4849..4871)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4860..4876
                     /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(4884..4890,4891..4908,4909..4914))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4929..4950
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5067..5084)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5238..5826)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5318..5337)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5997..6788,6789..6857))
                     /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     6620..6639
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6858..6962)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
       61 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc
      121 ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat
      181 gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt
      241 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
      301 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
      361 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
      421 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
      481 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
      541 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
      601 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
      661 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
      721 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
      781 agagctctcc ctatcagtga tagagatctc cctatcagtg atagagatcg tcgacgagct
      841 cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga
      901 agacaccggg accgatccag cctccggact ctagcgttta aacttaagct tcgccatgca
      961 tcatcatcac caccacagca gcggcgagga tgtcacgctc catatcgttg agcggccgta
     1021 ttccggattt cccgatgctt cctcagaggg cccggagccc acccaagggg aggcgcgggc
     1081 cacggaggag ccgtcgggga ccggctccga cgagctgatc aagtcggacc aggtgaacgg
     1141 tgtgctggtg ctgagccttc tggataaaat catcggcgcc gttgaccaga tccagctgac
     1201 ccaagcccag ctggaggagc gacaggcgga gatggagggc gctgtgcaga gcatccaggg
     1261 agagctgagc aagctgggca aggcgcacgc caccacgagc aacaccgtga gcaagttgct
     1321 ggagaaggtg cgcaaggtca gcgtcaacgt gaagaccgtg cgcggcagcc tggagcgcca
     1381 ggccggccag ataaagaaac tggaggtcaa cgaggcggag ctgctgaggc gccgcaactt
     1441 caaagtcatg atctaccagg atgaagtcaa gctgccggcc aaactgagcg tcagcaagtc
     1501 gctgaaagag tcggaggcac tgcctgagaa ggagggtgac gagctgggcg agggcgagcg
     1561 gcccgaggat gacaccgcgg cgatcgagct gtcgtccgac gaggcggtgg aggtggagga
     1621 ggtgatcgag gagtcccgcg ccgagcgcat caagcgcagc ggcctgcggc gcgtggacga
     1681 cttcaagaag gccttctcca aggagaagat ggagaagacc aaggtgcgca cgcgtgagaa
     1741 cctggagaag acgcgcctga agaccaagga gaacctggag aagacacggc acacgctgga
     1801 gaagcgcatg aacaagctgg gcacgcgcct ggtgcccgtg gagcgacgag agaagctgaa
     1861 gacatcccgg gacaagctgc gcaagtcctt cacgcccgac catgtggtgt atgcgcgctc
     1921 caagaccgct gtctacaagg tgccgccttt caccttccac gtcaagaaga tccgcgaggg
     1981 cgaggtggag gtgctgaagg ccaccgagat ggtggaggtg ggtcccgagg acgacgaggt
     2041 tggcgcggag cgcggcgagg ccactgacct gctgcgcggg agcagccccg acgtgcacac
     2101 gctgctggag atcaccgagg agtcggacgc cgtcctggtg gacaagagcg acagcgacgc
     2161 ggatccaccg gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     2221 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     2281 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     2341 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     2401 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     2461 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     2521 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     2581 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     2641 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     2701 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     2761 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
     2821 gaagcgcgat cacatggtcc ttaaggagtt cgtgaccgcc gccgggatca ctctcggcat
     2881 ggacgagctg tacaagtaaa gcggccgctc gagtctagag ggcccgttta aacccgctga
     2941 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     3001 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     3061 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     3121 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     3181 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     3241 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     3301 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     3361 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     3421 cccaaaaaac ttgattaggg tgatggttca cgtacctaga agttcctatt ccgaagttcc
     3481 tattctctag aaagtatagg aacttccttg gccaaaaagc ctgaactcac cgcgacgtct
     3541 gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag
     3601 ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta
     3661 aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc
     3721 gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc
     3781 atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct
     3841 gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg
     3901 agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc
     3961 atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc
     4021 agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa
     4081 gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc
     4141 ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc
     4201 aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag
     4261 cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt
     4321 cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag
     4381 ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc
     4441 cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac
     4501 cgacgcccca gcactcgtcc gagggcaaag gaatagcacg tactacgaga tttcgattcc
     4561 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     4621 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     4681 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     4741 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     4801 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     4861 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     4921 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     4981 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     5041 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     5101 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     5161 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     5221 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     5281 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     5341 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     5401 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     5461 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     5521 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     5581 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     5641 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     5701 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     5761 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     5821 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     5881 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     5941 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     6001 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     6061 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     6121 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     6181 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     6241 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     6301 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     6361 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     6421 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     6481 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     6541 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     6601 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     6661 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     6721 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     6781 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     6841 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     6901 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     6961 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc
     7021 tatgg
//
