LOCUS       sequence-356879-        7164 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TEVP-inducible tri-transmembrane SEAP RELEASE construct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7164)
  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 7164)
  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-3TM-tevs-KKMP
FEATURES             Location/Qualifiers
     source          1..7164
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        167..546
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        547..750
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     701..721
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    752..770
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    773..791
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     801..825
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             923..2425
                     /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             2651..2671
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2929..2958
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(3014..3031)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3020..3244
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3416..3435)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3567..3614
                     /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             3622..4644
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3622..4644
                     /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    4774..4895
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4811..4830)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4865..4884
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4944..4960)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4944..4960)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4957..4979)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4968..4984
                     /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(4992..4998,4999..5016,5017..5022))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5037..5058
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5175..5192)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5346..5934)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5426..5445)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6105..6896,6897..6965))
                     /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     6728..6747
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6966..7070)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7145..7164)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga
       61 gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta
      121 gggttaggcg ttttgcgctg cttcgtacgg gccagatata cgcgttgaca ttgattattg
      181 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
      241 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      301 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      361 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      421 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      481 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      541 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
      601 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
      661 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
      721 gtacggtggg aggtctatat aagcagagct ctccctatca gtgatagaga tctccctatc
      781 agtgatagag atcgtcgacg agctcgttta gtgaaccgtc agatcgcctg gagacgccat
      841 ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccg gactctagcg
      901 tttaaactta agcttcgcca ccatgctgct cctgttgctg ctgctgggcc tgaggctaca
      961 gctctccctg ggcatcatcc cagttgagga ggagaacccg gacttctgga accgcgaggc
     1021 agccgaggcc ctgggtgccg ccaagaagct gcagcctgca cagacagccg ccaagaacct
     1081 catcatcttc ctgggcgatg ggatgggggt gtctacggtg acagctgcca ggattctaaa
     1141 agggcagaag aaggacaaac tggggcctga gatacccctg gccatggacc gcttcccata
     1201 tgtggctctg tccaagacat acaatgtaga caaacatgtg ccagacagtg gagccacagc
     1261 cacggcctac ctgtgcgggg tcaagggcaa cttccagacc attggcttga gtgcagccgc
     1321 ccgctttaac cagtgcaaca cgacacgcgg caacgaggtc atctccgtga tgaatcgggc
     1381 caagaaagca gggaagtcag tgggagtggt aaccaccaca cgagtgcagc acgcctcgcc
     1441 agccggcacc tacgcccaca cggtgaaccg caactggtac tcggacgccg acgtgcctgc
     1501 ctcggcccgc caggaggggt gccaggacat cgctacgcag ctcatctcca acatggacat
     1561 tgacgtgatc ctaggtggag gccgaaaata catgtttccc atgggaaccc cagaccctga
     1621 gtacccagat gactacagcc aaggtgggac caggctggac gggaagaatc tggtgcagga
     1681 atggctggcg aagcgccagg gtgcccggta tgtgtggaac cgcactgagc tcatgcaggc
     1741 ttccctggac ccgtctgtga cccatctcat gggtctcttt gagcctggag acatgaaata
     1801 cgagatccac cgagactcca cactggaccc ctccctgatg gagatgacag aggctgccct
     1861 gcgcctgctg agcaggaacc cccgcggctt cttcctcttc gtggagggtg gtcgcatcga
     1921 ccatggtcat catgaaagca gggcttaccg ggcactgact gagacgatca tgttcgacga
     1981 cgccattgag agggcgggcc agctcaccag cgaggaggac acgctgagcc tcgtcactgc
     2041 cgaccactcc cacgtcttct ccttcggagg ctaccccctg cgagggagct ccatcttcgg
     2101 gctggcccct ggcaaggccc gggacaggaa ggcctacacg gtcctcctat acggaaacgg
     2161 tccaggctat gtgctcaagg acggcgctcg gccggatgtt accgagagcg agagcgggag
     2221 ccccgagtat cggcagcagt cagcagtgcc cctggacgaa gagacccacg caggcgagga
     2281 cgtggcggtg ttcgcgcgcg gcccgcaggc gcacctggtt cacggcgtgc aggagcagac
     2341 cttcatagcg cacgtcatgg ccttcgccgc ctgcctggag ccctacaccg cctgcgacct
     2401 ggcacccccc gccggcacca ccgacgccgc gcacccgggt ggatcctctg gcagaagcaa
     2461 aagaagcgtc attgacggct ttaccctgac cagcgacgag gtgtgggtgg tgggcatggg
     2521 catcgtcatg tctctcatcg tcctggccat cgtgtttggc aatgtgctgg tcatcacagc
     2581 cattgccaag ttcgagcgtc tgcagacggt caccgggggc ggctctctcg aggggggggg
     2641 ctcggggctc gaaaacttgt acttccagag tggcagtgga ggtaccaatt acttcatcac
     2701 ttcactggcc tgtgctgatc tggtcatggg cctggcagtg gtgccctttg gggccgccca
     2761 tattcttatg aaaatgtgga cttttggcaa cttctgggat atctacatct gggctccctt
     2821 ggccgggact tgtggggtcc ttctcctgtc actggttatc accctttact gcaacaagta
     2881 caaaagcaga cgcagtttta ttgatgaaaa gaaaatgcct tagggcccga acaaaaactc
     2941 atctcagaag aggatctgaa tagcgccgtc gaccatcatc ataatcatca ttgagtttaa
     3001 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     3061 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     3121 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     3181 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     3241 atggagatct atcgatgttc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     3301 accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg
     3361 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     3421 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     3481 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     3541 gattagggtg atggttcacg tacctagaag ttcctattcc gaagttccta ttctctagaa
     3601 agtataggaa cttccttggc caaaaagcct gaactcaccg cgacgtctgt cgagaagttt
     3661 ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct
     3721 cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc
     3781 gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt
     3841 ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat ctcccgccgt
     3901 gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg
     3961 gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc
     4021 ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt
     4081 gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc
     4141 gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc
     4201 gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc
     4261 attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc
     4321 tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg
     4381 gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc
     4441 tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac
     4501 gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg
     4561 gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc
     4621 actcgtccga gggcaaagga atagcacgta ctacgagatt tcgattccac cgccgccttc
     4681 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     4741 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     4801 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     4861 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     4921 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     4981 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     5041 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     5101 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     5161 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     5221 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     5281 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     5341 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     5401 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     5461 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     5521 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     5581 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     5641 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     5701 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     5761 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     5821 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     5881 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     5941 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     6001 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     6061 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     6121 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     6181 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     6241 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     6301 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     6361 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     6421 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     6481 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     6541 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     6601 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     6661 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     6721 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     6781 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     6841 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     6901 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     6961 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     7021 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     7081 cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc
     7141 tcagtacaat ctgctctgat gccg
//
