LOCUS       sequence-323514-        6730 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP tagged Tac in a RUSH vector in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6730)
  AUTHORS   Sun X, Tie HC, Chen B, Lu L
  TITLE     Glycans function as a Golgi export signal to promote the 
            constitutive exocytic trafficking.
  JOURNAL   J Biol Chem. 2020 Aug 21. pii: RA120.014476. doi: 
            10.1074/jbc.RA120.014476.
  PUBMED    32826314
REFERENCE   2  (bases 1 to 6730)
  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: "J Biol 
            Chem. 2020 Aug 21. pii: RA120.014476. doi: 
            10.1074/jbc.RA120.014476."
COMMENT     Sequence Label: SBP-GFP-Tac
FEATURES             Location/Qualifiers
     source          1..6730
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     intron          1646..1875
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     1858..1877
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(1947..2521,2522..2524,2525..2533)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2114..2131)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2341..2360
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     sig_peptide     2547..2606
                     /codon_start=1
                     /product="human interleukin-2 signal sequence"
                     /label=IL-2 signal sequence
                     /translation="MYRMQLLSCIALSLALVTNS"
     CDS             2607..2717
                     /codon_start=1
                     /product="streptavidin-binding peptide"
                     /label=SBP
                     /note="selected from a peptide library; binds streptavidin 
                     with nanomolar affinity (Keefe et al., 2001)"
                     /translation="DEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP"
     CDS             join(2727..2729,2730..2732,2733..3443)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2727..2729,2730..2732,2733..3443)
                     /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(2772..2793)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3033..3052)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3380..3401
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4350..4471
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4387..4406)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4573..4589)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4573..4589)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4586..4608)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4597..4613
                     /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(4621..4627,4628..4645,4646..4651))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4666..4687
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4804..4821)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4975..5563)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5055..5074)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5734..6525,6526..6594))
                     /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     6357..6376
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6595..6699)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcggat cgatatctgc ggcctagcta gcgcttaagg cctgttaacc ggtatggatg
      961 tatgcgtccg tcttgccctg tggctcctct ggggactcct cctgcaccag ggccagagcc
     1021 tcagccatag tcacagtgag aaggcgacag gaaccagctc gggcgccggt ttaaacgacc
     1081 ctagcaaaga ctcaaaagct caggtgtccg ctgccgaggc tggcattact ggaacatggt
     1141 acaatcagct cgggagcacc tttattgtga ctgctggagc cgatggagcc ctcaccggaa
     1201 catacgaatc tgctgtggga aacgccgaat cacggtacgt cctcactggc cgatacgata
     1261 gtgcccctgc caccgacgga tctgggactg ccctgggatg gactgtcgct tggaaaaaca
     1321 actaccggaa tgctcattct gccacaacat ggagtggaca gtacgtggga ggcgctgagg
     1381 ctagaatcaa tacacagtgg ctgctcacat ctggcacaac cgaggcaaat gcttggaaat
     1441 ccaccctggt gggacatgac acattcacca aagtgaaacc ctccgccgct tcaatcgatg
     1501 ccgccaaaaa agccggagtc aacaacggca atcctctgga tgccgtccag cagctcggac
     1561 cgaaagatga actgtgagcg gccgcataga taactgatcc agtgtgctgg aattaattcg
     1621 ctgtctgcga gggccagctg ttggggtgag tactccctct caaaagcggg catgacttct
     1681 gcgctaagat tgtcagtttc caaaaacgag gaggatttga tattcacctg gcccgcggtg
     1741 atgcctttga gggtggccgc gtccatctgg tcagaaaaga caatcttttt gttgtcaagc
     1801 ttgaggtgtg gcaggcttga gatctggcca tacacttgag tgacaatgac atccactttg
     1861 cctttctctc cacaggtgtc cactcccagg tccaactgca ggtcgagcat gcatctaggg
     1921 cggccaattc cgcccctctc cccccccccc tctccctccc ccccccctaa cgttactggc
     1981 cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg
     2041 ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct
     2101 aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca
     2161 gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg
     2221 aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct
     2281 gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa
     2341 tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt
     2401 atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa
     2461 aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa
     2521 gcttgccaca acccgggagg cgcgccatgt acaggatgca actcctgtct tgcattgcac
     2581 taagtcttgc acttgtcacg aattccgacg agaagaccac tggttggcga ggtggacacg
     2641 ttgttgaagg actggctggg gaacttgaac aacttcgtgc acgactggag catcacccac
     2701 aaggtcaacg tgaaccacct gcaggtatgg tgagcaaggg cgaggagctg ttcaccgggg
     2761 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     2821 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     2881 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     2941 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     3001 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     3061 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     3121 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     3181 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     3241 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     3301 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     3361 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     3421 tcggcatgga cgagctgtac aagggccggc cagacgagct ctgtgacgat gacccgccag
     3481 agatcccaca cgccacattc aaagccatgg cctacaagga aggaaccatg ttgaactgtg
     3541 aatgcaagag aggtttccgc agaataaaaa gcgggtcact ctatatgctc tgtacaggaa
     3601 actctagcca ctcgtcctgg gacaaccaat gtcaatgcac aagctctgcc actcggaaca
     3661 caacgaaaca agtgacacct caacctgaag aacagaaaga aaggaaaacc acagaaatgc
     3721 aaagtccaat gcagccagtg gaccaagcga gccttccagg tcactgcagg gaacctccac
     3781 catgggaaaa tgaagccaca gagagaattt atcatttcgt ggtggggcag atggtttatt
     3841 atcagtgcgt ccagggatac agggctctac acagaggtcc tgctgagagc gtctgcaaaa
     3901 tgacccacgg gaagacaagg tggacccagc cccagctcat atgcacaggt gaaatggaga
     3961 ccagtcagtt tccaggtgaa gagaagcctc aggcaagccc cgaaggccgt cctgagagtg
     4021 agacttcctg cctcgtcaca acaacagatt ttcaaataca gacagaaatg gctgcaacca
     4081 tggagacgtc catatttaca acagagtacc aggtagcagt ggccggctgt gttttcctgc
     4141 tgatcagcgt cctcctcctg agtgggctca cctggcagcg gagacagagg aagagtagaa
     4201 gaacaatcta gctcgagggc caattaatta actctagata actgatcata atcagccata
     4261 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga
     4321 aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca
     4381 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     4441 gtggtttgtc caaactcatc aatgtatctt aacgcgtcga gtgcattcta gttgtggttt
     4501 gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt
     4561 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     4621 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     4681 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     4741 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     4801 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     4861 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     4921 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     4981 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     5041 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     5101 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     5161 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     5221 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     5281 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     5341 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     5401 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     5461 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     5521 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     5581 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     5641 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     5701 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     5761 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     5821 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     5881 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     5941 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     6001 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     6061 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     6121 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     6181 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     6241 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     6301 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     6361 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     6421 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     6481 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     6541 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     6601 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     6661 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     6721 acctgacgtc
//
