LOCUS       sequence-357161-        6954 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TEVP-inducible single TM SEAP RELEASE construct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6954)
  AUTHORS   Vlahos AE, Kang J, Aldrete CA, Zhu R, Chong LS, Elowitz MB, Gao XJ
  TITLE     Protease-controlled secretion and display of intercellular signals.
  JOURNAL   Nat Commun. 2022 Feb 17;13(1):912. doi: 10.1038/s41467-022-28623-y.
  PUBMED    35177637
REFERENCE   2  (bases 1 to 6954)
  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: "Nat 
            Commun."; date: "2022-02-17"; volume: "13(1):912. doi"; pages: " 
            10.1038/s41467-022-28623-y"
COMMENT     Sequence Label: CMVTO-SEAP-26Sfur-B2AD-tevs-KKMP
FEATURES             Location/Qualifiers
     source          1..6954
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        131..510
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        511..714
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     665..685
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    716..734
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    737..755
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     765..789
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             887..2389
                     /codon_start=1
                     /product="secreted alkaline phosphatase from human 
                     placenta"
                     /label=SEAP
                     /note="The natural enzyme has a C-terminal transmembrane 
                     sequence that is replaced by a GPI anchor."
                     /translation="MLLLLLLLGLRLQLSLGIIPVEEENPDFWNREAAEALGAAKKLQP
                     AQTAAKNLIIFLGDGMGVSTVTAARILKGQKKDKLGPEIPLAMDRFPYVALSKTYNVDK
                     HVPDSGATATAYLCGVKGNFQTIGLSAAARFNQCNTTRGNEVISVMNRAKKAGKSVGVV
                     TTTRVQHASPAGTYAHTVNRNWYSDADVPASARQEGCQDIATQLISNMDIDVILGGGRK
                     YMFPMGTPDPEYPDDYSQGGTRLDGKNLVQEWLAKRQGARYVWNRTELMQASLDPSVTH
                     LMGLFEPGDMKYEIHRDSTLDPSLMEMTEAALRLLSRNPRGFFLFVEGGRIDHGHHESR
                     AYRALTETIMFDDAIERAGQLTSEEDTLSLVTADHSHVFSFGGYPLRGSSIFGLAPGKA
                     RDRKAYTVLLYGNGPGYVLKDGARPDVTESESGSPEYRQQSAVPLDEETHAGEDVAVFA
                     RGPQAHLVHGVQEQTFIAHVMAFAACLEPYTACDLAPPAGTTD"
     CDS             2594..2614
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2683..2712
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2768..2785)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2774..2998
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3170..3189)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3321..3368
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3376..4398
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3376..4398
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4528..4649
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4565..4584)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4619..4638
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4698..4714)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4698..4714)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4711..4733)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4722..4738
                     /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(4746..4752,4753..4770,4771..4776))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4791..4812
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4929..4946)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5100..5688)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5180..5199)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5859..6650,6651..6719))
                     /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     6482..6501
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6720..6824)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6899..6918)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg
       61 acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgt acgggccaga
      121 tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
      181 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
      241 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
      301 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
      361 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
      421 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
      481 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
      541 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
      601 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
      661 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctccct
      721 atcagtgata gagatctccc tatcagtgat agagatcgtc gacgagctcg tttagtgaac
      781 cgtcagatcg cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac
      841 cgatccagcc tccggactct agcgtttaaa cttaagcttc gccaccatgc tgctcctgtt
      901 gctgctgctg ggcctgaggc tacagctctc cctgggcatc atcccagttg aggaggagaa
      961 cccggacttc tggaaccgcg aggcagccga ggccctgggt gccgccaaga agctgcagcc
     1021 tgcacagaca gccgccaaga acctcatcat cttcctgggc gatgggatgg gggtgtctac
     1081 ggtgacagct gccaggattc taaaagggca gaagaaggac aaactggggc ctgagatacc
     1141 cctggccatg gaccgcttcc catatgtggc tctgtccaag acatacaatg tagacaaaca
     1201 tgtgccagac agtggagcca cagccacggc ctacctgtgc ggggtcaagg gcaacttcca
     1261 gaccattggc ttgagtgcag ccgcccgctt taaccagtgc aacacgacac gcggcaacga
     1321 ggtcatctcc gtgatgaatc gggccaagaa agcagggaag tcagtgggag tggtaaccac
     1381 cacacgagtg cagcacgcct cgccagccgg cacctacgcc cacacggtga accgcaactg
     1441 gtactcggac gccgacgtgc ctgcctcggc ccgccaggag gggtgccagg acatcgctac
     1501 gcagctcatc tccaacatgg acattgacgt gatcctaggt ggaggccgaa aatacatgtt
     1561 tcccatggga accccagacc ctgagtaccc agatgactac agccaaggtg ggaccaggct
     1621 ggacgggaag aatctggtgc aggaatggct ggcgaagcgc cagggtgccc ggtatgtgtg
     1681 gaaccgcact gagctcatgc aggcttccct ggacccgtct gtgacccatc tcatgggtct
     1741 ctttgagcct ggagacatga aatacgagat ccaccgagac tccacactgg acccctccct
     1801 gatggagatg acagaggctg ccctgcgcct gctgagcagg aacccccgcg gcttcttcct
     1861 cttcgtggag ggtggtcgca tcgaccatgg tcatcatgaa agcagggctt accgggcact
     1921 gactgagacg atcatgttcg acgacgccat tgagagggcg ggccagctca ccagcgagga
     1981 ggacacgctg agcctcgtca ctgccgacca ctcccacgtc ttctccttcg gaggctaccc
     2041 cctgcgaggg agctccatct tcgggctggc ccctggcaag gcccgggaca ggaaggccta
     2101 cacggtcctc ctatacggaa acggtccagg ctatgtgctc aaggacggcg ctcggccgga
     2161 tgttaccgag agcgagagcg ggagccccga gtatcggcag cagtcagcag tgcccctgga
     2221 cgaagagacc cacgcaggcg aggacgtggc ggtgttcgcg cgcggcccgc aggcgcacct
     2281 ggttcacggc gtgcaggagc agaccttcat agcgcacgtc atggccttcg ccgcctgcct
     2341 ggagccctac accgcctgcg acctggcacc ccccgccggc accaccgacg ccgcgcaccc
     2401 gggtggatcc tctggcagaa gcaaaagaag cgtcattgac ggctttaccc tgaccagcga
     2461 cgaggtgtgg gtggtgggca tgggcatcgt catgtctctc atcgtcctgg ccatcgtgtt
     2521 tggcaatgtg ctggtcatca cagccattgc caagttcgag cgtctgcaga cggtcaccgg
     2581 gggcggctct ctcgagaact tgtacttcca gagtggcagt ggaggtacca agtacaaaag
     2641 cagacgcagt tttattgatg aaaagaaaat gccttagggc ccgaacaaaa actcatctca
     2701 gaagaggatc tgaatagcgc cgtcgaccat catcataatc atcattgagt ttaaacccgc
     2761 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     2821 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     2881 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     2941 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggag
     3001 atctatcgat gttctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc
     3061 tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     3121 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     3181 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     3241 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     3301 ggtgatggtt cacgtaccta gaagttccta ttccgaagtt cctattctct agaaagtata
     3361 ggaacttcct tggccaaaaa gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc
     3421 gaaaagttcg acagcgtctc cgacctgatg cagctctcgg agggcgaaga atctcgtgct
     3481 ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg taaatagctg cgccgatggt
     3541 ttctacaaag atcgttatgt ttatcggcac tttgcatcgg ccgcgctccc gattccggaa
     3601 gtgcttgaca ttggggaatt cagcgagagc ctgacctatt gcatctcccg ccgtgcacag
     3661 ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg
     3721 gaggccatgg atgcgatcgc tgcggccgat cttagccaga cgagcgggtt cggcccattc
     3781 ggaccgcaag gaatcggtca atacactaca tggcgtgatt tcatatgcgc gattgctgat
     3841 ccccatgtgt atcactggca aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag
     3901 gctctcgatg agctgatgct ttgggccgag gactgccccg aagtccggca cctcgtgcac
     3961 gcggatttcg gctccaacaa tgtcctgacg gacaatggcc gcataacagc ggtcattgac
     4021 tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg ccaacatctt cttctggagg
     4081 ccgtggttgg cttgtatgga gcagcagacg cgctacttcg agcggaggca tccggagctt
     4141 gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg gtcttgacca actctatcag
     4201 agcttggttg acggcaattt cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc
     4261 gtccgatccg gagccgggac tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc
     4321 tggaccgatg gctgtgtaga agtactcgcc gatagtggaa accgacgccc cagcactcgt
     4381 ccgagggcaa aggaatagca cgtactacga gatttcgatt ccaccgccgc cttctatgaa
     4441 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     4501 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     4561 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     4621 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
     4681 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     4741 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     4801 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4861 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     4921 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     4981 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     5041 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     5101 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     5161 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     5221 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     5281 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     5341 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     5401 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     5461 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     5521 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     5581 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     5641 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     5701 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     5761 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     5821 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     5881 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     5941 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     6001 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     6061 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     6121 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     6181 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     6241 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     6301 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     6361 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     6421 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     6481 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     6541 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     6601 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     6661 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     6721 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     6781 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     6841 gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta
     6901 caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgt
//
