LOCUS       sequence-459269-        6012 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Membrane-bound nanoluciferase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6012)
  AUTHORS   Aldrete CA, Call CC, Sant'Anna LE, Vlahos AE, Pei J, Cong Q, Gao XJ
  TITLE     Orthogonalized human protease control of secreted signals.
  JOURNAL   Nat Chem Biol. 2025 Jan 15. doi: 10.1038/s41589-024-01831-x.
  PUBMED    39814991
REFERENCE   2  (bases 1 to 6012)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat Chem 
            Biol. 2025 Jan 15. doi: 10.1038/s41589-024-01831-x."
COMMENT     Sequence Label: CA-0194 CMVTO-secNLuc-rens-mem
FEATURES             Location/Qualifiers
     source          1..6012
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     265..284
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(503..607)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(682..701)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        868..1247
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1248..1451
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1402..1422
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    1453..1471
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1474..1492
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1502..1526
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1711..2220
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     CDS             2478..2507
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2563..2580)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2569..2793
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2965..2984)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3116..3163
                     /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             3171..4193
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3171..4193
                     /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    4323..4444
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4360..4379)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4414..4433
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4493..4509)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4493..4509)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4506..4528)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4517..4533
                     /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(4541..4547,4548..4565,4566..4571))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4586..4607
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4724..4741)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4895..5483)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4975..4994)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5654..6012,1..433,434..502))
                     /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"
ORIGIN
        1 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
       61 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
      121 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
      181 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
      241 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
      301 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
      361 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
      421 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
      481 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
      541 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
      601 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga
      661 tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct
      721 gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac
      781 aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct
      841 gcttcgtacg ggccagatat acgcgttgac attgattatt gactagttat taatagtaat
      901 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
      961 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
     1021 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
     1081 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
     1141 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     1201 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
     1261 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
     1321 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
     1381 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
     1441 taagcagagc tctccctatc agtgatagag atctccctat cagtgataga gatcgtcgac
     1501 gagctcgttt agtgaaccgt cagatcgcct ggagacgcca tccacgctgt tttgacctcc
     1561 atagaagaca ccgggaccga tccagcctcc ggactctagc gtttaaactt aagcttcgcc
     1621 accatgaact ccttctccac aagcgccttc ggtccagttg ccttctccct gggcctgctc
     1681 ctggtgttgc ctgctgcctt ccctgcccca gtcttcacac tcgaagattt cgttggggac
     1741 tggcgacaga cagccggcta caacctggac caagtccttg aacagggagg tgtgtccagt
     1801 ttgtttcaga atctcggggt gtccgtaact ccgatccaaa ggattgtcct gagcggtgaa
     1861 aatgggctga agatcgacat ccatgtcatc atcccgtatg aaggtctgag cggcgaccaa
     1921 atgggccaga tcgaaaaaat ttttaaggtg gtgtaccctg tggatgatca tcactttaag
     1981 gtgatcctgc actatggcac actggtaatc gacggggtta cgccgaacat gatcgactat
     2041 ttcggacggc cgtatgaagg catcgccgtg ttcgacggca aaaagatcac tgtaacaggg
     2101 accctgtgga acggcaacaa aattatcgac gagcgcctga tcaaccccga cggctccctg
     2161 ctgttccgag taaccatcaa cggagtgacc ggctggcggc tgtgcgaacg cattctggcg
     2221 tatattcacc cattccatct ggttattcat aacgagtcgg gcgccgggac aggagggtct
     2281 gggggtggca ctggcggatc tggaggagac tacaaggacg atgacgacaa cggcggatcg
     2341 ggggggtctg gtggtagtgg cggttcgggg ggaggcacag gtggatccct ggtcgtggtt
     2401 gctggagtcc tgttttgtta tggcttgcta gtgacagtgg ctctttgtgt tgaaaacggg
     2461 ggcggctctt agggcccgaa caaaaactca tctcagaaga ggatctgaat agcgccgtcg
     2521 accatcatca taatcatcat tgagtttaaa cccgctgatc agcctcgact gtgccttcta
     2581 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     2641 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     2701 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     2761 gcaggcatgc tggggatgcg gtgggctcta tggagatcta tcgatgttct ggggatgcgg
     2821 tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg
     2881 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     2941 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     3001 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     3061 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt acctagaagt
     3121 tcctattccg aagttcctat tctctagaaa gtataggaac ttccttggcc aaaaagcctg
     3181 aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc
     3241 tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg
     3301 gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgtttatc
     3361 ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg gaattcagcg
     3421 agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg
     3481 aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg
     3541 ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca
     3601 ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg
     3661 tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg
     3721 ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc
     3781 tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt
     3841 cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt atggagcagc
     3901 agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt
     3961 atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg
     4021 atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc gggactgtcg
     4081 ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt gtagaagtac
     4141 tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa tagcacgtac
     4201 tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     4261 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     4321 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4381 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     4441 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     4501 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     4561 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     4621 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     4681 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     4741 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     4801 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     4861 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     4921 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     4981 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5041 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     5101 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     5161 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     5221 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     5281 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     5341 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     5401 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     5461 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     5521 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     5581 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     5641 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     5701 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     5761 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     5821 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     5881 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     5941 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     6001 gtatggcttc at
//
