LOCUS       sequence-335212-        6859 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a sfGFP dropout expression cassette in place of Parts 1-5 to
            be used in the Spike Display system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6859)
  AUTHORS   Javanmardi K, Chou CW, Terrace CI, Annapareddy A, Kaoud TS, Guo Q, 
            Lutgens J, Zorkic H, Horton AP, Gardner EC, Nguyen G, Boutz DR, 
            Goike J, Voss WN, Kuo HC, Dalby KN, Gollihar JD, Finkelstein IJ
  TITLE     Rapid characterization of spike variants via mammalian cell surface 
            display.
  JOURNAL   Mol Cell. 2021 Dec 16;81(24):5099-5111.e8. doi: 
            10.1016/j.molcel.2021.11.024.
  PUBMED    34919820
REFERENCE   2  (bases 1 to 6859)
  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: 
            "10.1016/j.molcel.2021.11"; journalName: "Mol Cell"; date: 
            "2021-12-16- 16"; volume: "81"; issue: "24"; pages: "5099-5111.e8"
COMMENT     Sequence Label: Spike Display_Part 1-5 DO
FEATURES             Location/Qualifiers
     source          1..6859
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..64)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    53..277
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(410..429)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    561..608
                     /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             616..1638
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             616..1638
                     /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    1768..1889
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1805..1824)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1859..1878
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1938..1954)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1938..1954)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1951..1973)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1962..1978
                     /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(1986..1992,1993..2010,2011..2016))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2031..2052
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2169..2186)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2340..2928)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2420..2439)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3099..3890,3891..3959))
                     /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     3722..3741
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3960..4064)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4139..4158)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4330..4709
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4710..4913
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4864..4884
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    4915..4933
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4936..4954
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     4964..4988
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          5107..5239
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     5222..5241
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        5284..5302
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     sig_peptide     5348..5407
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     RBS             5570..5581
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             5588..6301
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      6313..6384
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6400..6427
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             6545..6610
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             6617..6640
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             6647..6670
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             6713..6859
                     /codon_start=1
                     /product="transmembrane domain from platelet derived growth
                     factor receptor beta"
                     /label=PDGFR-beta TM domain
                     /translation="AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQ
                     KKPR"
ORIGIN
        1 taacggccgc tcgagtctag agggcccgtt taaacccgct gatcagcctc gactgtgcct
       61 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
      121 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
      181 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
      241 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc
      301 tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
      361 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
      421 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
      481 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
      541 ggtgatggtt cacgtaccta gaagttccta ttccgaagtt cctattctct agaaagtata
      601 ggaacttcct tggccaaaaa gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc
      661 gaaaagttcg acagcgtctc cgacctgatg cagctctcgg agggcgaaga atctcgtgct
      721 ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg taaatagctg cgccgatggt
      781 ttctacaaag atcgttatgt ttatcggcac tttgcatcgg ccgcgctccc gattccggaa
      841 gtgcttgaca ttggggaatt cagcgagagc ctgacctatt gcatctcccg ccgtgcacag
      901 ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg
      961 gaggccatgg atgcgatcgc tgcggccgat cttagccaga cgagcgggtt cggcccattc
     1021 ggaccgcaag gaatcggtca atacactaca tggcgtgatt tcatatgcgc gattgctgat
     1081 ccccatgtgt atcactggca aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag
     1141 gctctcgatg agctgatgct ttgggccgag gactgccccg aagtccggca cctcgtgcac
     1201 gcggatttcg gctccaacaa tgtcctgacg gacaatggcc gcataacagc ggtcattgac
     1261 tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg ccaacatctt cttctggagg
     1321 ccgtggttgg cttgtatgga gcagcagacg cgctacttcg agcggaggca tccggagctt
     1381 gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg gtcttgacca actctatcag
     1441 agcttggttg acggcaattt cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc
     1501 gtccgatccg gagccgggac tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc
     1561 tggaccgatg gctgtgtaga agtactcgcc gatagtggaa accgacgccc cagcactcgt
     1621 ccgagggcaa aggaatagca cgtactacga gatttcgatt ccaccgccgc cttctatgaa
     1681 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     1741 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     1801 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     1861 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
     1921 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     1981 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     2041 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     2101 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     2161 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     2221 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     2281 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     2341 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     2401 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     2461 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     2521 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     2581 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     2641 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     2701 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     2761 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     2821 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     2881 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     2941 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     3001 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     3061 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     3121 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     3181 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     3241 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     3301 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     3361 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     3421 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     3481 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     3541 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     3601 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     3661 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     3721 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     3781 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     3841 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     3901 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     3961 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     4021 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     4081 gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta
     4141 caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg
     4201 tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt
     4261 gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat
     4321 atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag
     4381 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
     4441 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
     4501 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
     4561 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
     4621 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
     4681 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
     4741 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
     4801 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
     4861 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctcccta
     4921 tcagtgatag agatctccct atcagtgata gagatcgtcg acgagctcgt ttagtgaacc
     4981 gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga caccgggacc
     5041 gatccagcct ccggactcta gcgtttaaac ttaagctgca gaagttggtc gtgaggcact
     5101 gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa actgggcttg
     5161 tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac tgacatccac
     5221 tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta aggctagagt
     5281 acttaatacg actcactata ggctagcctc gagaattcac gcgtggtacc tctagagtcg
     5341 acccgggatg gagacagaca cactcctgct atgggtactg ctgctctggg ttccaggttc
     5401 cactggtgac ggtggcaggt ggaaagtgaa acgtgatttc atgcgtcatt ttgaacattt
     5461 tgtaaatctt atttaataat gtgtgcggca attcacattt aatttatgaa tgttttctta
     5521 acatcgcggc aactcaagaa acggcaggtt cggatcttag ctactagaga aagaggagaa
     5581 atactagatg cgtaaaggcg aagagctgtt cactggtgtc gtccctattc tggtggaact
     5641 ggatggtgat gtcaacggtc ataagttttc cgtgcgtggc gagggtgaag gtgacgcaac
     5701 taatggtaaa ctgacgctga agttcatctg tactactggt aaactgccgg ttccttggcc
     5761 gactctggta acgacgctga cttatggtgt tcagtgcttt gctcgttatc cggaccatat
     5821 gaagcagcat gacttcttca agtccgccat gccggaaggc tatgtgcagg aacgcacgat
     5881 ttcctttaag gatgacggca cgtacaaaac gcgtgcggaa gtgaaatttg aaggcgatac
     5941 cctggtaaac cgcattgagc tgaaaggcat tgactttaaa gaggacggca atatcctggg
     6001 ccataagctg gaatacaatt ttaacagcca caatgtttac atcaccgccg ataaacaaaa
     6061 aaatggcatt aaagcgaatt ttaaaattcg ccacaacgtg gaggatggca gcgtgcagct
     6121 ggctgatcac taccagcaaa acactccaat cggtgatggt cctgttctgc tgccagacaa
     6181 tcactatctg agcacgcaaa gcgttctgtc taaagatccg aacgagaaac gcgatcatat
     6241 ggttctgctg gagttcgtaa ccgcagcggg catcacgcat ggtatggatg aactgtacaa
     6301 atgaccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc
     6361 tgttgtttgt cggtgaacgc tctctactag agtcacactg gctcaccttc gggtgggcct
     6421 ttctgcgttt atacacctgc caccgcaggg atccggatac atccccgagg cccccagaga
     6481 tggccaggcc tacgtgcgga aggacggcga gtgggtactg ctgagcacat tcctgggcag
     6541 atccgactac aaagaccatg acggtgatta taaagatcat gacatcgatt acaaggatga
     6601 cgatgacaag agcgcctggt cccaccccca gttcgagaag ggatccctgg aggtgctgtt
     6661 ccagggccca ggcggcggcg gtagtggagg gggcggatct ggcggctcag ctgctgtggg
     6721 ccaggacacg caggaggtca tcgtggtgcc acactccttg ccctttaagg tggtggtgat
     6781 ctcagccatc ctggccctgg tggtgctcac catcatctcc cttatcatcc tcatcatgct
     6841 ttggcagaag aagccacgt
//
