LOCUS       sequence-457428-        5606 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express human PHOSPHO2 protein (C-terminal Tobacco Etch Virus (TEV) 
            protease cleavable Twin-Strep-fusion protein 
            (ENLYFQGS-WSHPQFEK-(GGGS)2-GGSA-WSHPQFEK)) in mammalian cell.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5606)
  TITLE     PHOSPHO2 is PA phosphatase and PHOSPHO1 is PLC
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5606)
  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            "Unpublished"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: pCAGGS-PHOSPHO2-TEV-Twin-Strep
FEATURES             Location/Qualifiers
     source          1..5606
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          663..1679
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1603..1625
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1687..1706
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2507..2527
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(2537..2560,2561..2596,2597..2620)
                     /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="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     primer_bind     complement(2627..2646)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2692..2747
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2746..2765)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3108..3124)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3108..3124)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3121..3143)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3132..3148
                     /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(3156..3162,3163..3180,3181..3186))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3201..3222
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3281..3476
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3327..3462
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3482..3616
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3519..3538)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3573..3592
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3684..3701)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3855..4443)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3935..3954)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4614..5405,5406..5474))
                     /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     5237..5256
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5475..5579)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 ttgtcgactt cccattgccc gtgggacggc tgagcccccg ttcgcccccc cccccccccc
      421 tccccccccc cccccccccc ccattttttt tttttttttt tttttaattt ttgggggggg
      481 gggggggggg gggggggggg ggggggcggg cgccgggggg gggggggcgg ggcgaggggc
      541 ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt
      601 ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg
      661 cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc
      721 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
      781 ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg tggctgcgtg
      841 aaagccttga ggggctccgg gagggccctt tgtgcggggg gagcggctcg gggggtgcgt
      901 gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc gctgcccggc ggctgtgagc
      961 gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt gcgcgagggg agcgcggccg
     1021 ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa caaaggctgc gtgcggggtg
     1081 tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt cgggctgcaa ccccccctgc
     1141 acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc cgtacggggc
     1201 gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg ccgggcgggg
     1261 cggggccgcc tcgggccggg gagggctcgg gggaggggcg cggcggcccc cggagcgccg
     1321 gcggctgtcg aggcgcggcg agccgcagcc attgcctttt atggtaatcg tgcgagaggg
     1381 cgcagggact tcctttgtcc caaatctgtg cggagccgaa atctgggagg cgccgccgca
     1441 ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg
     1501 agggccttcg tgcgtcgccg cgccgccgtc cccttctccc tctccagcct cggggctgtc
     1561 cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg
     1621 tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc
     1681 tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa ttgagctccc
     1741 gggtaccgaa ttcgatatcg cggccgctcg agccaccatg aaaattttgc tagtttttga
     1801 ctttgacaat acaatcatag atgacaatag tgacacttgg attgtacaat gtgctcccaa
     1861 caaaaagctt cctattgaac tacgtgattc ttatcgaaaa ggattttgga cagaatttat
     1921 gggcagagtc tttaagtatt tgggagataa gggtgtaaga gaacatgaaa tgaaaagagc
     1981 agtgacatca ttgcctttca ctccagggat ggtggaactc ttcaacttta taagaaagaa
     2041 taaggataaa tttgactgca ttattatttc agattcaaat tcggtcttca tagattgggt
     2101 tttagaagct gccagttttc atgacatatt tgataaagtg tttacaaatc cagcagcttt
     2161 taatagcaat ggtcatctca ctgttgaaaa ttatcatact cattcttgca atagatgccc
     2221 aaagaatctt tgcaaaaagg tagttttgat agaatttgta gataaacagt tacaacaggg
     2281 agtgaattat acacaaattg tttatattgg tgatggtgga aatgatgtct gtccagtcac
     2341 ctttttaaag aatgatgatg ttgccatgcc acggaaagga tataccttac agaaaactct
     2401 ttccagaatg tctcaaaatc ttgagcctat ggaatattct gttgtagttt ggtcctcagg
     2461 tgttgatata atttctcatt tacaatttct aataaaggat ctcgaggaaa acctctattt
     2521 tcaaggcgcc accatgtggt ctcatcccca gttcgaaaaa ggtggtggaa gtggtggggg
     2581 ctcaggagga tcagcctggt cacaccctca gttcgagaag tgaagatctt tttccctctg
     2641 ccaaaaatta tggggacatc atgaagcccc ttgagcatct gacttctggc taataaagga
     2701 aatttatttt cattgcaata gtgtgttgga attttttgtg tctctcactc ggaaggacat
     2761 atgggagggc aaatcattta aaacatcaga atgagtattt ggtttagagt ttggcaacat
     2821 atgcccatat gctggctgcc atgaacaaag gttggctata aagaggtcat cagtatatga
     2881 aacagccccc tgctgtccat tccttattcc atagaaaagc cttgacttga ggttagattt
     2941 tttttatatt ttgttttgtg ttattttttt ctttaacatc cctaaaattt tccttacatg
     3001 ttttactagc cagatttttc ctcctctcct gactactccc agtcatagct gtccctcttc
     3061 tcttatggag atccctcgac ctgcagccca agcttggcgt aatcatggtc atagctgttt
     3121 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     3181 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     3241 cccgctttcc agtcgggaaa cctgtcgtgc cagcggatcc gcatctcaat tagtcagcaa
     3301 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     3361 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     3421 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     3481 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     3541 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     3601 ttatcatgtc tggatccgct gcattaatga atcggccaac gcgcggggag aggcggtttg
     3661 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     3721 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     3781 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     3841 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     3901 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     3961 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4021 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     4081 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     4141 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4201 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     4261 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     4321 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4381 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     4441 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     4501 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     4561 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     4621 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     4681 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     4741 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     4801 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     4861 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     4921 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     4981 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     5041 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     5101 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     5161 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     5221 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     5281 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     5341 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     5401 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     5461 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     5521 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     5581 acatttcccc gaaaagtgcc acctgg
//
