LOCUS       sequence-356901-        6963 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HCVP-inducible single TM SEAP RELEASE construct with GS linkers.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6963)
  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 6963)
  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-hcvs-KKMP (GS)
FEATURES             Location/Qualifiers
     source          1..6963
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        81..460
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        461..664
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     615..635
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    666..684
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    687..705
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     715..739
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             837..2339
                     /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             2642..2671
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2727..2744)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2733..2957
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3129..3148)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3280..3327
                     /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             3335..4357
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3335..4357
                     /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    4487..4608
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4524..4543)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4578..4597
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4657..4673)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4657..4673)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4670..4692)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4681..4697
                     /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(4705..4711,4712..4729,4730..4735))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4750..4771
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4888..4905)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5059..5647)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5139..5158)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5818..6609,6610..6678))
                     /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     6441..6460
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6679..6783)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6858..6877)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgt
       61 acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac
      121 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg
      181 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
      241 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
      301 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
      361 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
      421 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta
      481 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga
      541 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa
      601 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag
      661 agctctccct atcagtgata gagatctccc tatcagtgat agagatcgtc gacgagctcg
      721 tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc tccatagaag
      781 acaccgggac cgatccagcc tccggactct agcgtttaaa cttaagcttc gccaccatgc
      841 tgctcctgtt gctgctgctg ggcctgaggc tacagctctc cctgggcatc atcccagttg
      901 aggaggagaa cccggacttc tggaaccgcg aggcagccga ggccctgggt gccgccaaga
      961 agctgcagcc tgcacagaca gccgccaaga acctcatcat cttcctgggc gatgggatgg
     1021 gggtgtctac ggtgacagct gccaggattc taaaagggca gaagaaggac aaactggggc
     1081 ctgagatacc cctggccatg gaccgcttcc catatgtggc tctgtccaag acatacaatg
     1141 tagacaaaca tgtgccagac agtggagcca cagccacggc ctacctgtgc ggggtcaagg
     1201 gcaacttcca gaccattggc ttgagtgcag ccgcccgctt taaccagtgc aacacgacac
     1261 gcggcaacga ggtcatctcc gtgatgaatc gggccaagaa agcagggaag tcagtgggag
     1321 tggtaaccac cacacgagtg cagcacgcct cgccagccgg cacctacgcc cacacggtga
     1381 accgcaactg gtactcggac gccgacgtgc ctgcctcggc ccgccaggag gggtgccagg
     1441 acatcgctac gcagctcatc tccaacatgg acattgacgt gatcctaggt ggaggccgaa
     1501 aatacatgtt tcccatggga accccagacc ctgagtaccc agatgactac agccaaggtg
     1561 ggaccaggct ggacgggaag aatctggtgc aggaatggct ggcgaagcgc cagggtgccc
     1621 ggtatgtgtg gaaccgcact gagctcatgc aggcttccct ggacccgtct gtgacccatc
     1681 tcatgggtct ctttgagcct ggagacatga aatacgagat ccaccgagac tccacactgg
     1741 acccctccct gatggagatg acagaggctg ccctgcgcct gctgagcagg aacccccgcg
     1801 gcttcttcct cttcgtggag ggtggtcgca tcgaccatgg tcatcatgaa agcagggctt
     1861 accgggcact gactgagacg atcatgttcg acgacgccat tgagagggcg ggccagctca
     1921 ccagcgagga ggacacgctg agcctcgtca ctgccgacca ctcccacgtc ttctccttcg
     1981 gaggctaccc cctgcgaggg agctccatct tcgggctggc ccctggcaag gcccgggaca
     2041 ggaaggccta cacggtcctc ctatacggaa acggtccagg ctatgtgctc aaggacggcg
     2101 ctcggccgga tgttaccgag agcgagagcg ggagccccga gtatcggcag cagtcagcag
     2161 tgcccctgga cgaagagacc cacgcaggcg aggacgtggc ggtgttcgcg cgcggcccgc
     2221 aggcgcacct ggttcacggc gtgcaggagc agaccttcat agcgcacgtc atggccttcg
     2281 ccgcctgcct ggagccctac accgcctgcg acctggcacc ccccgccggc accaccgacg
     2341 ccgcgcaccc gggtggatcc tctggcagaa gcaaaagaag cgtcattgac ggctttaccc
     2401 tgaccagcga cgaggtgtgg gtggtgggca tgggcatcgt catgtctctc atcgtcctgg
     2461 ccatcgtgtt tggcaatgtg ctggtcatca cagccattgc caagttcgag cgtctgcaga
     2521 cggtcaccgg gggcggctct ctcgaggatg tcgtctgctg ctcaatgtcc tacggcagtg
     2581 gaggtaccaa gtacaaaagc agacgcagtt ttattgatga aaagaaaatg ccttagggcc
     2641 cgaacaaaaa ctcatctcag aagaggatct gaatagcgcc gtcgaccatc atcataatca
     2701 tcattgagtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     2761 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     2821 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     2881 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     2941 tgcggtgggc tctatggaga tctatcgatg ttctggggat gcggtgggct ctatggcttc
     3001 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     3061 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     3121 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     3181 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     3241 ccccaaaaaa cttgattagg gtgatggttc acgtacctag aagttcctat tccgaagttc
     3301 ctattctcta gaaagtatag gaacttcctt ggccaaaaag cctgaactca ccgcgacgtc
     3361 tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc gacctgatgc agctctcgga
     3421 gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt
     3481 aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc
     3541 cgcgctcccg attccggaag tgcttgacat tggggaattc agcgagagcc tgacctattg
     3601 catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc
     3661 tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc ttagccagac
     3721 gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt
     3781 catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt
     3841 cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga
     3901 agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg
     3961 cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc
     4021 caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga
     4081 gcggaggcat ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg
     4141 tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca
     4201 gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc
     4261 ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa
     4321 ccgacgcccc agcactcgtc cgagggcaaa ggaatagcac gtactacgag atttcgattc
     4381 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     4441 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
     4501 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     4561 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
     4621 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     4681 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     4741 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     4801 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4861 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     4921 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     4981 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     5041 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     5101 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     5161 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     5221 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     5281 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     5341 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     5401 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     5461 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     5521 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5581 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5641 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     5701 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     5761 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     5821 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     5881 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     5941 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     6001 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     6061 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     6121 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     6181 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     6241 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     6301 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     6361 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     6421 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     6481 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     6541 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     6601 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     6661 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     6721 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     6781 gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc
     6841 ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc
     6901 cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg
     6961 caa
//
