LOCUS       sequence-417735-        6101 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial overexpression vector for C-terminally Strep and His 
            tagged mScarlet fluorescent protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6101)
  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 6101)
  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: pET28a-mScarlet-TEV-Strep-His
FEATURES             Location/Qualifiers
     source          1..6101
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    13..152
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(78..97)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     238..260
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(254..445)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1254..2336)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     2298..2317
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2337..2414)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2620..2639
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2723..2741
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2742..2766
                     /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)."
     RBS             2781..2803
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3512..3532
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(3545..3550,3551..3574,3575..3610,3611..3634)
                     /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             3650..3679
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(3758..3776)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3772..3819
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3856..4311
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3943..3962)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4153..4174
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4403..5218)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(4403..5218)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5126..5145
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5210..5229
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5340..5928
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5829..5848
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6082..6099
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgtggta tttcacaccg
       61 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
      121 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
      181 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
      241 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
      301 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
      361 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
      421 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
      481 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
      541 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
      601 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
      661 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
      721 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
      781 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
      841 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
      901 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
      961 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     1021 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     1081 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     1141 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     1201 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     1261 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     1321 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     1381 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     1441 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     1501 ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac
     1561 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     1621 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     1681 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     1741 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     1801 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     1861 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     1921 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     1981 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     2041 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     2101 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     2161 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     2221 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     2281 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     2341 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     2401 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     2461 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     2521 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     2581 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     2641 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
     2701 cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac
     2761 aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacca tggtcagcaa
     2821 aggggaagcc gtcattaaag aattcatgcg cttcaaagtc catatggagg gatccatgaa
     2881 cggccacgag ttcgagatcg agggtgaggg ggagggccgg ccctacgagg ggacacaaac
     2941 tgccaagctg aaagtgacaa agggcggacc attgcctttt tcttgggata tcttgagccc
     3001 ccagttcatg tacgggtcaa gggcatttac taagcacccg gctgatatcc ctgattacta
     3061 taagcagtct ttcccagaag gcttcaaatg ggaacgggtc atgaactttg aggacggcgg
     3121 cgcggtgacc gtcactcagg acaccagcct tgaagatggt acactcattt acaaggttaa
     3181 gctgagaggc acaaacttcc cacctgatgg gccggtgatg caaaaaaaga ccatgggctg
     3241 ggaggccagc accgagaggc tctatcccga agatggggtc ctcaaaggag atatcaaaat
     3301 ggctctgcgc ctcaaagatg gcggcaggta tctcgcagac ttcaaaacaa cctacaaggc
     3361 caagaaaccc gtccagatgc ccggcgctta caacgttgat cgaaaattgg atattaccag
     3421 tcacaacgag gattacactg tggtggagca gtacgaaaga agcgagggaa ggcatagcac
     3481 tggtgggatg gacgagctgt acaaactcga ggagaatttg tactttcaag gcggtggagg
     3541 agggagtgcc tggtctcatc cacagtttga aaagggagga ggatccggag gtggtagtgg
     3601 cggatctgca tggtcccacc ctcagtttga gaagggcggc ggaggttctc atcaccacca
     3661 ccatcatcat caccaccact aatctagaac tatagtgagt cgtattacga tccggctgct
     3721 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     3781 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     3841 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     3901 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     3961 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     4021 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     4081 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     4141 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     4201 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     4261 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     4321 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     4381 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
     4441 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     4501 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     4561 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     4621 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
     4681 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     4741 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag
     4801 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     4861 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     4921 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     4981 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     5041 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     5101 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     5161 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa
     5221 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct
     5281 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     5341 tgaaatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     5401 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     5461 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     5521 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     5581 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     5641 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     5701 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     5761 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     5821 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     5881 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     5941 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     6001 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     6061 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg a
//
