LOCUS       sequence-457427-        5753 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express human PHOSPHO1 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 5753)
  TITLE     PHOSPHO2 is PA phosphatase and PHOSPHO1 is PLC
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5753)
  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            "Unpublished"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-PHOSPHO1-TEV-Twin-Strep
FEATURES             Location/Qualifiers
     source          1..5753
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          732..1748
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1672..1694
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1756..1775
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2654..2674
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(2684..2707,2708..2743,2744..2767)
                     /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(2774..2793)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2839..2894
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2893..2912)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3255..3271)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3255..3271)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3268..3290)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3279..3295
                     /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(3303..3309,3310..3327,3328..3333))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3348..3369
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3428..3623
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3474..3609
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3629..3763
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3666..3685)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3720..3739
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3831..3848)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4002..4590)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4082..4101)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4761..5552,5553..5621))
                     /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     5384..5403
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5622..5726)
                     /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 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
      421 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
      481 cgatgggggc gggggggggg ggggggggcg cgccaggggg gggggggggg gggggggggg
      541 gggggggggg gggggggggg gggggggggg gggggcgcgc gccaggcggg gcggggcggg
      601 gcgaggggcg gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc
      661 tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg
      721 cgcggcgggc gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct
      781 cgcgccgccc gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg
      841 gcccttctcc tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt
      901 ggctgcgtga aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg
      961 ggggtgcgtg cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg
     1021 gctgtgagcg ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga
     1081 gcgcggccgg gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg
     1141 tgcggggtgt gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac
     1201 cccccctgca cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc
     1261 gtacggggcg tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc
     1321 cgggcggggc ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc
     1381 ggagcgccgg cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt
     1441 gcgagagggc gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc
     1501 gccgccgcac cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa
     1561 tgggcgggga gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc
     1621 ggggctgtcc gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct
     1681 tctggcgtgt gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt
     1741 tcctacagct cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat
     1801 tgagctcccg ggtaccgaat tcgatatcgc ggccgctcga gccaccatga gtggctgttt
     1861 tccagtttct ggcctccgct gcctatctag ggacggcagg atggccgcgc agggcgcgcc
     1921 gcgcttcctc ctgaccttcg acttcgacga gactatcgtg gacgaaaaca gcgacgattc
     1981 gatcgtgcgc gccgcgccgg gccagcggct cccggagagc ctgcgagcca cctaccgcga
     2041 gggcttctac aacgagtaca tgcagcgcgt cttcaagtac ctgggcgagc agggcgtgcg
     2101 gccgcgggac ctgagcgcca tctacgaagc catccctttg tcgccaggca tgagcgacct
     2161 gctgcagttt gtggcaaaac agggcgcctg cttcgaggtg attctcatct ccgatgccaa
     2221 cacctttggc gtggagagct cgctgcgcgc cgccggccac cacagcctgt tccgccgcat
     2281 cctcagcaac ccgtcggggc cggatgcgcg gggactgctg gctctgcggc cgttccacac
     2341 acacagctgc gcgcgctgcc ccgccaacat gtgcaagcac aaggtgctca gcgactacct
     2401 gcgcgagcgg gcccacgacg gcgtgcactt cgagcgcctc ttctacgtgg gcgacggcgc
     2461 caacgacttc tgccccatgg ggctgctggc gggcggcgac gtggccttcc cgcgccgcgg
     2521 ctaccccatg caccgcctca ttcaggaggc ccagaaggcc gagcccagct cgttccgcgc
     2581 cagcgtggtg ccctgggaaa cggctgcaga tgtgcgcctc cacctgcaac aggtgctgaa
     2641 gtcgtgcctc gaggaaaacc tctattttca aggcgccacc atgtggtctc atccccagtt
     2701 cgaaaaaggt ggtggaagtg gtgggggctc aggaggatca gcctggtcac accctcagtt
     2761 cgagaagtga agatcttttt ccctctgcca aaaattatgg ggacatcatg aagccccttg
     2821 agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt
     2881 ttttgtgtct ctcactcgga aggacatatg ggagggcaaa tcatttaaaa catcagaatg
     2941 agtatttggt ttagagtttg gcaacatatg cccatatgct ggctgccatg aacaaaggtt
     3001 ggctataaag aggtcatcag tatatgaaac agccccctgc tgtccattcc ttattccata
     3061 gaaaagcctt gacttgaggt tagatttttt ttatattttg ttttgtgtta tttttttctt
     3121 taacatccct aaaattttcc ttacatgttt tactagccag atttttcctc ctctcctgac
     3181 tactcccagt catagctgtc cctcttctct tatggagatc cctcgacctg cagcccaagc
     3241 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     3301 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     3361 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     3421 cggatccgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     3481 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     3541 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt
     3601 ttggaggcct aggcttttgc aaaaagctaa cttgtttatt gcagcttata atggttacaa
     3661 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     3721 tggtttgtcc aaactcatca atgtatctta tcatgtctgg atccgctgca ttaatgaatc
     3781 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     3841 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     3901 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     3961 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4021 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     4081 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     4141 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     4201 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     4261 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4321 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     4381 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     4441 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     4501 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     4561 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     4621 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     4681 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     4741 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     4801 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     4861 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     4921 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     4981 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     5041 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     5101 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     5161 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     5221 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     5281 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     5341 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     5401 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     5461 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     5521 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     5581 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     5641 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     5701 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgg
//
