LOCUS       sequence-163489-        4713 bp    DNA     circular SYN 15-DEC-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 4713)
  TITLE     Barr-Gillespie TwinStrep Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4713)
  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: 
            "Unpublished"
COMMENT     Sequence Label: CAPZA1-TwinStrepII
FEATURES             Location/Qualifiers
     source          1..4713
                     /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(1691..1696,1697..1720,1721..1756,1757..1780)
                     /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(1784..1808)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1837..1958
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1848..1867)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1902..1921
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2206..2223)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2377..2965)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2457..2476)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3136..3927,3928..3996))
                     /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     3759..3778
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3997..4101)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4128..4583
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4215..4234)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4425..4446
                     /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 tggccgactt
      841 tgaggatcgg gtgtcggatg aagagaaggt ccgcatagcc gctaaattca tcacccatgc
      901 acccccaggg gagtttaatg aagtattcaa tgatgtccgg ctactgctta acaatgacaa
      961 tctcctcagg gaaggggcag cacatgcatt tgcccagtat aacatggatc agttcacacc
     1021 cgtgaagata gaaggctacg atgaccaggt cttaattaca gagcatggtg accttggtaa
     1081 cagcagattt ttagatccaa gaaaccaaat ttcctttaag tttgatcatt tacggaaaga
     1141 agcaagtgac ccgcagccag aggatgtgga tggaggcttg aagtcttgga gggagtcgtg
     1201 tgacagtgct ctgagagcct atgtgaaaga tcattattcc aacggcttct gtactgttta
     1261 cgctaaaact atagatgggc aacagaccat tattgcatgt attgaaagcc accagtttca
     1321 accaaaaaac ttctggaatg gtcgctggag atcagagtgg aagttcacca ttacaccacc
     1381 ttcagcccag gtggttggag tactcaagat tcaggttcat tattatgaag atggcaatgt
     1441 tcagctggtt agtcataaag atgtgcagga ctcagtaact gtttctaatg aaatccaaac
     1501 taccaaggag tttattaaaa tcatagagag tgcagaaaat gagtatcaga cagcaattag
     1561 tgaaaattac caaactatgt cagacaccac gttcaaggcc ttgcgccggc agcttccagt
     1621 tacccgcacc aaaatcgact ggaacaagat actcagttac aagattggca aagaaatgca
     1681 gaatgctaga tctgcatggt cccatccgca atttgagaag gggggcggat ctggaggggg
     1741 cagtggaggt tccgcttgga gccaccccca gttcgaaaag tgaacccagc tttcttgtac
     1801 aaagtggtga tggccggccg cttcgagcag acatgataag atacattgat gagtttggac
     1861 aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
     1921 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
     1981 ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca
     2041 aatgtggtaa aatcgataag gatccgtcga ccgatgccct tgagagcctt caacccagtc
     2101 agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt
     2161 atcatgcaac tcgtaggaca ggtgccggca gcgctcttcc gcttcctcgc tcactgactc
     2221 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     2281 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     2341 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     2401 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     2461 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     2521 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     2581 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     2641 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     2701 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     2761 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     2821 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     2881 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     2941 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     3001 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     3061 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     3121 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     3181 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     3241 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     3301 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     3361 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     3421 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     3481 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     3541 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     3601 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     3661 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     3721 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     3781 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     3841 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     3901 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     3961 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     4021 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     4081 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg cgccctgtag
     4141 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     4201 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     4261 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     4321 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     4381 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     4441 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     4501 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     4561 caaaatatta acgcttacaa tttgccattc gccattcagg ctgcgcaact gttgggaagg
     4621 gcgatcggtg cgggcctctt cgctattacg ccagcccaag ctaccatgat aagtaagtaa
     4681 tattaaggta cgggaggtac ttggagcggc cgc
//
