LOCUS       sequence-162200-        4803 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expression of tagged protein in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4803)
  TITLE     Barr-Gillespie TwinStrep Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4803)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: SLC9A3R2-TwinStrepII
FEATURES             Location/Qualifiers
     source          1..4803
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    1..49
                     /note="synthetic polyadenylation signal"
     misc_feature    63..154
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     103..122
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     protein_bind    190..210
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     enhancer        271..574
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        575..778
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     729..749
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    799..823
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      824..833
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1781..1786,1787..1810,1811..1846,1847..1870)
                     /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"
     protein_bind    complement(1874..1898)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1927..2048
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1938..1957)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1992..2011
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2296..2313)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2467..3055)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2547..2566)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3226..4017,4018..4086))
                     /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     3849..3868
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4087..4191)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4218..4673
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4305..4324)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4515..4536
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 aataaaatat ctttattttc attacatctg tgtgttggtt ttttgtgtga atcgatagta
       61 ctaacatacg ctctccatca aaacaaaacg aaacaaaaca aactagcaaa ataggctgtc
      121 cccagtgcaa gtgcaggtgc cagaacattt ctctatcgat aggtaccgag ctcttacgcg
      181 tgctagcatc aactttgtat agaaaagttg tagttattaa tagtaatcaa ttacggggtc
      241 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
      301 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
      361 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
      421 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
      481 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
      541 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
      601 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
      661 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
      721 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg
      781 tttagtgaac cgtcagatcc aagtttgtac aaaaaagcag ggcgccacca tggccgcgct
      841 ggaatcgctg cggccccgct tgtgtcgcct ggtgcgcgga gagcaagggt acggctttca
      901 cctgcatggc gagaaaggcc gccgtggcca gttcatccgg cgcgtggagc caggctcccc
      961 agccgaggcg gccgcgctgc gcgccgggga ccgcctggtt gaggtcaacg gcgtcaacgt
     1021 agagggcgag acgcatcacc aggtggtaca gaggatcaag gctgtggagg ggcagaccca
     1081 gctgcttgtg gtggacaagg agaccgatga ggagctctgc cggcggcaac tgacctgcac
     1141 tgaggagatg gcccatcgag ggcttccccc agcccacaac ccctgggagc caaagccgga
     1201 ctgggcatgc tcgggcagcc ttggctcgga cactggccag aaggatgtca atgggccccc
     1261 aagagagctg cgtccccggc tctgccactt gcgaagggga ccccagggct acgggttcaa
     1321 cctgcatagt gacaagtccc ggcctggcca gtacatccgc tctgtggacc ctggctctcc
     1381 tgcttctcac tctggcctcc gtgcccagga tcggctcatt gaggtgaatg ggcagaacgt
     1441 ggaggggctg cgacatgccg aggttgtcgc cagaatcaag gcacaggagg acgaggcccg
     1501 gctgctggta gtcgatcctg agactgatga acacttcaag cggttgaggg tcatacccac
     1561 tgaggaacat gtagaaggtc cactgccatc accagtcacg aacgggacca gtcctgccca
     1621 ggatggcagt acctggaagc gagacccctt ccaggaaagt ggcctccacc tgagtcccac
     1681 agcagctgag gccaaggaga aggctcgagc tactcgggtc aacaagcggg caccacagat
     1741 ggactggaac cggaagcgag agatcttcag caacttcaga tctgcatggt cccatccgca
     1801 atttgagaag gggggcggat ctggaggggg cagtggaggt tccgcttgga gccaccccca
     1861 gttcgaaaag tgaacccagc tttcttgtac aaagtggtga tggccggccg cttcgagcag
     1921 acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat
     1981 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
     2041 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg
     2101 aggtttttta aagcaagtaa aacctctaca aatgtggtaa aatcgataag gatccgtcga
     2161 ccgatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact
     2221 atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca
     2281 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     2341 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     2401 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     2461 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     2521 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     2581 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     2641 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     2701 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     2761 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     2821 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     2881 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     2941 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     3001 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     3061 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     3121 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     3181 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     3241 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     3301 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     3361 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     3421 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     3481 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     3541 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     3601 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     3661 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     3721 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     3781 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     3841 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     3901 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     3961 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     4021 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     4081 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     4141 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     4201 ccgaaaagtg ccacctgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     4261 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     4321 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     4381 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     4441 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     4501 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     4561 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     4621 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttgccattc
     4681 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg
     4741 ccagcccaag ctaccatgat aagtaagtaa tattaaggta cgggaggtac ttggagcggc
     4801 cgc
//
