LOCUS       sequence-158286-        4671 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 4671)
  TITLE     Barr-Gillespie TwinStrep Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4671)
  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            "Unpublished"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: CAPZB2-TwinStrepII
FEATURES             Location/Qualifiers
     source          1..4671
                     /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"
     CDS             join(1649..1654,1655..1678,1679..1714,1715..1738)
                     /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(1742..1766)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1795..1916
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1806..1825)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1860..1879
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2164..2181)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2335..2923)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2415..2434)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3094..3885,3886..3954))
                     /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     3717..3736
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3955..4059)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4086..4541
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4173..4192)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4383..4404
                     /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 tgagcgatca
      841 gcagctggac tgcgccttgg acctgatgag gcgcctgcct ccacagcaga ttgagaagaa
      901 cctcagcgat ctgatcgacc tggtccccag tctgtgtgaa gatctcctgt catctgttga
      961 ccagcccctg aaaattgcca gagacaaggt ggtgggcaag gattaccttt tgtgtgacta
     1021 caacagagac ggggactcct ataggtcacc gtggagtaac aagtatgacc ctcctttgga
     1081 agatggggcc atgccatctg ctcggctcag aaagctggag gtagaggcca acaatgcctt
     1141 cgaccaatac cgagacctgt attttgaagg tggggtctca tcagtctacc tctgggatct
     1201 tgatcatggc tttgctggag tgatcctcat aaagaaagct ggagatggat ccaagaagat
     1261 caaaggctgc tgggattcca tccacgtggt ggaagtgcag gagaagtcca gcggccgtac
     1321 tgcccattac aagttgacct ccacggtgat gctatggctg caaaccaaca aatccggctc
     1381 gggcaccatg aacctgggag gcagcctaac cagacagatg gagaaagacg aaactgtgag
     1441 tgactgttcc ccacacatag ccaacatcgg gcgcctggtg gaggacatgg aaaacaaaat
     1501 ccgaagcacg ctgaatgaga tctactttgg aaaaacaaag gacatcgtca acgggctgag
     1561 gtctgtgcag acgtttgcag acaaatcaaa gcaagaagcg cttaagaacg acctggtgga
     1621 ggccttgaag agaaagcagc agtgtagatc tgcatggtcc catccgcaat ttgagaaggg
     1681 gggcggatct ggagggggca gtggaggttc cgcttggagc cacccccagt tcgaaaagtg
     1741 aacccagctt tcttgtacaa agtggtgatg gccggccgct tcgagcagac atgataagat
     1801 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     1861 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
     1921 acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa
     1981 gcaagtaaaa cctctacaaa tgtggtaaaa tcgataagga tccgtcgacc gatgcccttg
     2041 agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca
     2101 cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc gctcttccgc
     2161 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     2221 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     2281 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     2341 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     2401 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     2461 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     2521 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     2581 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     2641 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     2701 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     2761 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     2821 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     2881 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     2941 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     3001 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     3061 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     3121 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     3181 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     3241 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     3301 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     3361 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     3421 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     3481 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     3541 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     3601 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     3661 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     3721 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     3781 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     3841 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     3901 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     3961 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     4021 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     4081 acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     4141 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     4201 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     4261 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     4321 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     4381 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     4441 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     4501 atttaacgcg aattttaaca aaatattaac gcttacaatt tgccattcgc cattcaggct
     4561 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agcccaagct
     4621 accatgataa gtaagtaata ttaaggtacg ggaggtactt ggagcggccg c
//
