LOCUS       sequence-357153-        6103 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Constitutively secreted GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6103)
  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 6103)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-HA-GFP
FEATURES             Location/Qualifiers
     source          1..6103
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        134..513
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        514..717
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     668..688
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    719..737
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    740..758
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     768..792
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(1097..1099,1100..1102,1103..1813)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             1835..1864
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1920..1937)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1926..2150
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2322..2341)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2473..2520
                     /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             2528..3550
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2528..3550
                     /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    3680..3801
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3717..3736)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3771..3790
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3850..3866)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3850..3866)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3863..3885)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3874..3890
                     /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(3898..3904,3905..3922,3923..3928))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3943..3964
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4081..4098)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4252..4840)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4332..4351)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5011..5802,5803..5871))
                     /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     5634..5653
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5872..5976)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6051..6070)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga
       61 ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgtacgggcc
      121 agatatacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca
      181 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      241 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      301 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      361 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      421 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      481 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      541 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      601 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      661 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc
      721 cctatcagtg atagagatct ccctatcagt gatagagatc gtcgacgagc tcgtttagtg
      781 aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg
      841 gaccgatcca gcctccggac tctagcgttt aaacttaagc ttcgccacca tgaagaccat
      901 cattgccctg agctacatct tctgcctggt gttcgctctc gaaatacggg cggcgtttct
      961 tcgccgaaga aatacggctc ttagaactag ggtagcagaa ctgagacaac gagtgcagcg
     1021 cctccgaaat atagtatcac agtatgaaac tcgctatggt ccgctcggcg ccgggacagg
     1081 agggtctggg ggtggcatgg tctcaaaggg cgaggaactt tttacgggcg ttgttccgat
     1141 cctcgtcgaa ttggacgggg acgttaatgg gcataaattc tcagttagtg gtgagggtga
     1201 gggggacgcg acatatggga aacttacgct gaagttcata tgtaccaccg gaaaattgcc
     1261 agtgccgtgg cctactctcg tcaccaccct gacatatggt gttcagtgtt ttagccggta
     1321 tccagaccat atgaaacagc acgatttttt taaatccgct atgcctgaag gttacgtaca
     1381 ggagcgaacg atatttttta aagacgacgg aaactacaaa actagagccg aggtaaaatt
     1441 cgagggtgac actctcgtga accggatcga actgaagggc atagatttta aggaggatgg
     1501 caatatactg ggccacaagc tcgaatacaa ctataattct cataatgtct atatcatggc
     1561 ggacaaacag aaaaacggca tcaaggtaaa ctttaaaatc cgacacaata ttgaggacgg
     1621 ctctgtccag cttgcggacc attaccaaca gaacacgccg ataggggatg gtccagtact
     1681 tctccctgat aatcactatt tgtctactca gtcagctctt agcaaggacc caaacgaaaa
     1741 acgggatcac atggtccttc ttgagttcgt cactgcggct gggataacac tcgggatgga
     1801 cgaactgtat aagtaggccg ctcgaggagg gcccgaacaa aaactcatct cagaagagga
     1861 tctgaatagc gccgtcgacc atcatcataa tcatcattga gtttaaaccc gctgatcagc
     1921 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     1981 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     2041 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     2101 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg agatctatcg
     2161 atgttctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     2221 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     2281 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     2341 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     2401 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     2461 ttcacgtacc tagaagttcc tattccgaag ttcctattct ctagaaagta taggaacttc
     2521 cttggccaaa aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt
     2581 cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt
     2641 cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa
     2701 agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga
     2761 cattggggaa ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac
     2821 gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat
     2881 ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca
     2941 aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt
     3001 gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga
     3061 tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt
     3121 cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga
     3181 ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt
     3241 ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc
     3301 gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt
     3361 tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc
     3421 cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga
     3481 tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc
     3541 aaaggaatag cacgtactac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     3601 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     3661 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     3721 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     3781 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     3841 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     3901 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     3961 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4021 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     4081 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     4141 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     4201 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     4261 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     4321 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     4381 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     4441 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     4501 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     4561 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     4621 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     4681 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     4741 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     4801 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     4861 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     4921 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     4981 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     5041 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     5101 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     5161 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     5221 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     5281 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     5341 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     5401 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     5461 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     5521 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     5581 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     5641 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     5701 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     5761 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     5821 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     5881 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     5941 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     6001 tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc
     6061 tctgatgccg catagttaag ccagtatctg ctccctgctt gtg
//
