LOCUS       sequence-468945-        8232 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For production of Extracellular Vesicles (EVs), with the receptor 
            Smoothened (Smo), Twin-Strep-tag (TST), and an HA tag at its 
            N-terminus on their surface.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8232)
  AUTHORS   Gonzalez-Magaldi M, Gullapalli A, Papoulas O, Liu C, Leung AY, Guo 
            L, Brilot A, Marcotte EM, Ke Z, Leahy DJ
  TITLE     Structure and organization of full-length Epidermal Growth Factor 
            Receptor in extracellular vesicles by cryo-electron tomography.
  JOURNAL   bioRxiv [Preprint]. 2024 Nov 28:2024.11.25.625301. doi: 
            10.1101/2024.11.25.625301.
  PUBMED    39651119
REFERENCE   2  (bases 1 to 8232)
  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: "bioRxiv 
            [Preprint]. 2024 Nov 28:2024.11.25.625301. doi: 
            10.1101/2024.11.25.625301."
COMMENT     Sequence Label: pHA TST 3C SMO EABR
FEATURES             Location/Qualifiers
     source          1..8232
                     /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"
     CDS             1025..1051
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(1064..1069,1070..1093,1094..1129,1130..1153)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             1160..1183
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(3823..3840)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3829..4053
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4099..4527
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4186..4205)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4396..4417
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4536..4556)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4541..4870
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4721..4856
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4783..4802
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4937..5731
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWVQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     5601..5620
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5905..6026
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5942..5961)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5996..6015
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6075..6091)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6075..6091)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6088..6110)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6099..6115
                     /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(6123..6129,6130..6147,6148..6153))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6168..6189
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6306..6323)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6477..7065)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6557..6576)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7236..8027,8028..8096))
                     /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     7859..7878
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8097..8201)
                     /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 gctggctagc
      901 gtttaaactt aagcttggta ccggatcctc tagagccacc atgcgaccct ccgggacggc
      961 cggggcagcg ctcctggcgc tgctggctgc gctctgcccg gcgagtcggg ctggcggagg
     1021 cagctaccca tacgacgtac cagattacgc tggaggaggc tccagtgcgt ggagtcatcc
     1081 tcaattcgag aaaggtggag gttctggcgg tggatcggga ggttcagcgt ggagccaccc
     1141 gcagttcgaa aaatccggac ttgaagtcct ctttcaggga cccggcgggg gctcctcgag
     1201 cgggaacgcg accgggcctg ggcctcggag cgcgggcggg agcgcgagga ggagcgcggc
     1261 ggtgactggc cctccgccgc cgctgagcca ctgcggccgg gctgccccct gcgagccgct
     1321 gcgctacaac gtgtgcctgg gctcggtgct gccctacggg gccacctcca cactgctggc
     1381 cggagactcg gactcccagg aggaagcgca cggcaagctc gtgctctggt cgggcctccg
     1441 gaatgccccc cgctgctggg cagtgatcca gcccctgctg tgtgccgtat acatgcccaa
     1501 gtgtgagaat gaccgggtgg agctgcccag ccgtaccctc tgccaggcca cccgaggccc
     1561 ctgtgccatc gtggagaggg agcggggctg gcctgacttc ctgcgctgca ctcctgaccg
     1621 cttccctgaa ggctgcacga atgaggtgca gaacatcaag ttcaacagtt caggccagtg
     1681 cgaagtgccc ttggttcgga cagacaaccc caagagctgg tacgaggacg tggagggctg
     1741 cggcatccag tgccagaacc cgctcttcac agaggctgag caccaggaca tgcacagcta
     1801 catcgcggcc ttcggggccg tcacgggcct ctgcacgctc ttcaccctgg ccacattcgt
     1861 ggctgactgg cggaactcga atcgctaccc tgctgttatt ctcttctacg tcaatgcgtg
     1921 cttctttgtg ggcagcattg gctggctggc ccagttcatg gatggtgccc gccgagagat
     1981 cgtctgccgt gcagatggca ccatgaggct tggggagccc acctccaatg agactctgtc
     2041 ctgcgtcatc atctttgtca tcgtgtacta cgccctgatg gctggtgtgg tttggtttgt
     2101 ggtcctcacc tatgcctggc acacttcctt caaagccctg ggcaccacct accagcctct
     2161 ctcgggcaag acctcctact tccacctgct cacctggtca ctcccctttg tcctcactgt
     2221 ggcaatcctt gctgtggcgc aggtggatgg ggactctgtg agtggcattt gttttgtggg
     2281 ctacaagaac taccgatacc gtgcgggctt cgtgctggcc ccaatcggcc tggtgctcat
     2341 cgtgggaggc tacttcctca tccgaggagt catgactctg ttctccatca agagcaacca
     2401 ccccgggctg ctgagtgaga aggctgccag caagatcaac gagaccatgc tgcgcctggg
     2461 catttttggc ttcctggcct ttggctttgt gctcattacc ttcagctgcc acttctacga
     2521 cttcttcaac caggctgagt gggagcgcag cttccgggac tatgtgctat gtcaggccaa
     2581 tgtgaccatc gggctgccca ccaagcagcc catccctgac tgtgagatca agaatcgccc
     2641 gagccttctg gtggagaaga tcaacctgtt tgccatgttt ggaactggca tcgccatgag
     2701 cacctgggtc tggaccaagg ccacgctgct catctggagg cgtacctggt gcaggttgac
     2761 tgggcagagt gacgatgagc caaagcggat caagaagagc aagatgattg ccaaggcctt
     2821 ctctaagcgg cacgagctcc tgcagaaccc aggccaggag ctgtccttca gcatgcacac
     2881 tgtgtcccac gacgggcccg tggcgggctt ggcctttgac ctcaatgagc cctcagctga
     2941 tgtctcctct gcctgggccc agcatgtcac caagatggtg gctcggagag gagccatact
     3001 gccccaggat atttctgtca cccctgtggc aactccagtg cccccagagg aacaagccaa
     3061 cctgtggctg gttgaggcag agatctcccc agagctgcag aagcgcctgg gccggaagaa
     3121 gaagaggagg aagaggaaga aggaggtgtg cccgctggcg ccgccccctg agcttcaccc
     3181 ccctgcccct gcccccagta ccattcctcg actgcctcag ctgccccggc agaaatgcct
     3241 ggtggctgca ggtgcctggg gagctgggga ctcttgccga cagggagcgt ggaccctggt
     3301 ctccaaccca ttctgcccag agcccagtcc ccctcaggat ccatttctgc ccagtgcacc
     3361 ggcccccgtg gcatgggctc atggccgccg acagggcctg gggcctattc actcccgcac
     3421 caacctgatg gacacagaac tcatggatgc agactcggac ttcgctttgc ctggcaatcc
     3481 cgatcacagg gaaatggggg aaactctccc agaagaggtt ggagaatatc ggcagccatc
     3541 tggtggcagt gtacctgtgt ctccgggtcc gcccagtggt cttgaaccca ctagctcctc
     3601 accttacggc ggcggctcct ttaacagctc aatcaacaac atccacgaaa tggagattca
     3661 gctgaaggat gcactcgaaa agaatcaaca atggctcgtt tacgaccaac aacgcgaggt
     3721 gtacgtgaaa ggattgcttg caaaaatttt cgaactggaa aagaaaacag agactgcagc
     3781 acatagtctt ccttgaaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta
     3841 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     3901 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     3961 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     4021 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
     4081 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     4141 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     4201 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     4261 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     4321 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     4381 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     4441 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     4501 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg
     4561 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     4621 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     4681 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     4741 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     4801 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     4861 cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg
     4921 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     4981 ggtggagagg ctattcggct atgactgggt acaacagaca atcggctgct ctgatgccgc
     5041 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     5101 tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     5161 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     5221 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     5281 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     5341 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     5401 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     5461 ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     5521 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     5581 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     5641 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     5701 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     5761 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     5821 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     5881 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     5941 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     6001 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     6061 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     6121 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     6181 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     6241 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     6301 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     6361 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     6421 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     6481 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     6541 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     6601 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     6661 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     6721 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     6781 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     6841 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     6901 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     6961 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     7021 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     7081 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     7141 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     7201 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     7261 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     7321 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     7381 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     7441 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     7501 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     7561 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     7621 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     7681 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     7741 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     7801 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     7861 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     7921 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     7981 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     8041 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     8101 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     8161 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     8221 ccacctgacg tc
//
