LOCUS       sequence-417733-        6814 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian overexpression vector for C-terminally TwinStrep and His 
            tagged human Prom1s1 (W795R).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6814)
  AUTHORS   Bell TA, Luce BE, Hakim P, Ananda VY, Dardari H, Nguyen TH, 
            Monshizadeh A, Chao LH
  TITLE     Prominin 1 and Tweety Homology 1 both induce extracellular vesicle 
            formation.
  JOURNAL   Elife. 2024 Aug 13;13:e100061. doi: 10.7554/eLife.100061.
  PUBMED    39168849
REFERENCE   2  (bases 1 to 6814)
  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: "Elife."; 
            date: "2024-08-13"; volume: "13:e100061. doi"; pages: " 
            10.7554/eLife.100061"
COMMENT     Sequence Label: pCS2-HsProm1s1(W795R)-TwinStrep-His
FEATURES             Location/Qualifiers
     source          1..6814
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             2666..2686
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(2699..2704,2705..2728,2729..2764,2765..2788)
                     /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             2804..2833
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     polyA_signal    2866..3000
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2890..2909)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2944..2963
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3113..3133)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3113..3131)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3152..3168)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3152..3168)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3165..3187)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3176..3192
                     /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(3200..3206,3207..3224,3225..3230))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3245..3266
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3383..3400)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3554..4142)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3634..3653)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4313..5104,5105..5173))
                     /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     4936..4955
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5174..5278)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5304..5759)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5441..5462)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5653..5672
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5830..6814
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atcccatcga tatggccctc gtactcggct ccctgttgct
      121 gctggggctg tgcgggaact ccttttcagg agggcagcct tcatccacag atgctcctaa
      181 ggcttggaat tatgaattgc ctgcaacaaa ttatgagacc caagactccc ataaagctgg
      241 acccattggc attctctttg aactagtgca tatctttctc tatgtggtac agccgcgtga
      301 tttcccagaa gatactttga gaaaattctt acagaaggca tatgaatcca aaattgatta
      361 tgacaagatt gtctactatg aagcagggat tattctatgc tgtgtcctgg ggctgctgtt
      421 tattattctg atgcctctgg tggggtattt cttttgtatg tgtcgttgct gtaacaaatg
      481 tggtggagaa atgcaccagc gacagaagga aaatgggccc ttcctgagga aatgctttgc
      541 aatctccctg ttggtgattt gtataataat aagcattggc atcttctatg gttttgtggc
      601 aaatcaccag gtaagaaccc ggatcaaaag gagtcggaaa ctggcagata gcaatttcaa
      661 ggacttgcga actctcttga atgaaactcc agagcaaatc aaatatatat tggcccagta
      721 caacactacc aaggacaagg cgttcacaga tctgaacagt atcaattcag tgctaggagg
      781 cggaattctt gaccgactga gacccaacat catccctgtt cttgatgaga ttaagtccat
      841 ggcaacagcg atcaaggaga ccaaagaggc gttggagaac atgaacagca ccttgaagag
      901 cttgcaccaa caaagtacac agcttagcag cagtctgacc agcgtgaaaa ctagcctgcg
      961 gtcatctctc aatgaccctc tgtgcttggt gcatccatca agtgaaacct gcaacagcat
     1021 cagattgtct ctaagccagc tgaatagcaa ccctgaactg aggcagcttc cacccgtgga
     1081 tgcagaactt gacaacgtta ataacgttct taggacagat ttggatggcc tggtccaaca
     1141 gggctatcaa tcccttaatg atatacctga cagagtacaa cgccaaacca cgactgtcgt
     1201 agcaggtatc aaaagggtct tgaattccat tggttcagat atcgacaatg taactcagcg
     1261 tcttcctatt caggatatac tctcagcatt ctctgtttat gttaataaca ctgaaagtta
     1321 catccacaga aatttaccta cattggaaga gtatgattca tactggtggc tgggtggcct
     1381 ggtcatctgc tctctgctga ccctcatcgt gattttttac tacctgggct tactgtgtgg
     1441 cgtgtgcggc tatgacaggc atgccacccc gaccacccga ggctgtgtct ccaacaccgg
     1501 aggcgtcttc ctcatggttg gagttggatt aagtttcctc ttttgctgga tattgatgat
     1561 cattgtggtt cttacctttg tctttggtgc aaatgtggaa aaactgatct gtgaacctta
     1621 cacgagcaag gaattattcc gggttttgga tacaccctac ttactaaatg aagactggga
     1681 atactatctc tctgggaagc tatttaataa atcaaaaatg aagctcactt ttgaacaagt
     1741 ttacagtgac tgcaaaaaaa atagaggcac ttacggcact cttcacctgc agaacagctt
     1801 caatatcagt gaacatctca acattaatga gcatactgga agcataagca gtgaattgga
     1861 aagtctgaag gtaaatctta atatctttct gttgggtgca gcaggaagaa aaaaccttca
     1921 ggattttgct gcttgtggaa tagacagaat gaattatgac agctacttgg ctcagactgg
     1981 taaatccccc gcaggagtga atcttttatc atttgcatat gatctagaag caaaagcaaa
     2041 cagtttgccc ccaggaaatt tgaggaactc cctgaaaaga gatgcacaaa ctattaaaac
     2101 aattcaccag caacgagtcc ttcctataga acaatcactg agcactctat accaaagcgt
     2161 caagatactt caacgcacag ggaatggatt gttggagaga gtaactagga ttctagcttc
     2221 tctggatttt gctcagaact tcatcacaaa caatacttcc tctgttatta ttgaggaaac
     2281 taagaagtat gggagaacaa taataggata ttttgaacat tatctgcagt ggatcgagtt
     2341 ctctatcagt gagaaagtgg catcgtgcaa acctgtggcc accgctctag atactgctgt
     2401 tgatgtcttt ctgtgtagct acattatcga ccccttgaat ttgtttcgtt ttggcatagg
     2461 aaaagctact gtatttttac ttccggctct aatttttgcg gtaaaactgg ctaagtacta
     2521 tcgtcgaatg gattcggagg acgtgtacga tgatgttgaa actataccca tgaaaaatat
     2581 ggaaaatggt aataatggtt atcataaaga tcatgtatat ggtattcaca atcctgttat
     2641 gacaagccca tcacaacatc tcgaggagaa tttgtacttt caaggcggtg gaggagggag
     2701 tgcctggtct catccacagt ttgaaaaggg aggaggatcc ggaggtggta gtggcggatc
     2761 tgcatggtcc caccctcagt ttgagaaggg cggcggaggt tctcatcacc accaccatca
     2821 tcatcaccac cactaatcta gaactatagt gagtcgtatt acgtagatcc agacatgata
     2881 agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt
     2941 tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt
     3001 aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt
     3061 taattcgcgg ccgcggcgcc aatgcattgg gcccggtacc cagcttttgt tccctttagt
     3121 gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     3181 atccgctcac aattccacac aacatacgag ccgggagcat aaagtgtaaa gcctggggtg
     3241 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     3301 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     3361 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     3421 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     3481 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     3541 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     3601 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     3661 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     3721 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     3781 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     3841 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     3901 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     3961 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     4021 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     4081 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4141 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     4201 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     4261 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     4321 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     4381 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     4441 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     4501 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     4561 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     4621 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     4681 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     4741 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     4801 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     4861 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     4921 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     4981 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     5041 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     5101 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     5161 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     5221 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     5281 catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt
     5341 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     5401 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     5461 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     5521 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     5581 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     5641 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     5701 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     5761 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     5821 attacgccag tcgaccgcca attcaatatg gcgtatatgg actcatgcca attcaatatg
     5881 gtggatctgg acctgtgcca attcaatatg gcgtatatgg actcgtgcca attcaatatg
     5941 gtggatctgg accccagcca attcaatatg gcggacttgg caccatgcca attcaatatg
     6001 gcggacctgg cactgtgcca actggggagg ggtctacttg gcacggtgcc aagtttgagg
     6061 aggggtcttg gccctgtgcc aagtccgcca tattgaattg gcatggtgcc aataatggcg
     6121 gccatattgg ctatatgcca ggatcaatat ataggcaata tccaatatgg ccctatgcca
     6181 atatggctat tggccaggtt caatactatg tattggccct atgccatata gtattccata
     6241 tatgggtttt cctattgacg tagatagccc ctcccaatgg gcggtcccat ataccatata
     6301 tggggcttcc taataccgcc catagccact cccccattga cgtcaatggt ctctatatat
     6361 ggtctttcct attgacgtca tatgggcggt cctattgacg tatatggcgc ctcccccatt
     6421 gacgtcaatt acggtaaatg gcccgcctgg ctcaatgccc attgacgtca ataggaccac
     6481 ccaccattga cgtcaatggg atggctcatt gcccattcat atccgttctc acgcccccta
     6541 ttgacgtcaa tgacggtaaa tggcccactt ggcagtacat caatatctat taatagtaac
     6601 ttggcaagta cattactatt ggaagtacgc cagggtacat tggcagtact cccattgacg
     6661 tcaatggcgg taaatggccc gcgatggctg ccaagtacat ccccattgac gtcaatgggg
     6721 aggggcaatg acgcaaatgg gcgttccatt gacgtaaatg ggcggtaggc gtgcctaatg
     6781 ggaggtctat ataagcaatg ctcgtttagg gaac
//
