LOCUS       Exported                8036 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  TIP-LITer2.0 gene circuit with the following architecture: 
            CMV-TetOx2-TetR-LOV-TIP-P2A-GFP.
ACCESSION   .
VERSION     .
KEYWORDS    pMTG05-Amp-FRT
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8036)
  AUTHORS   Guinn MT, Balazsi G
  TITLE     Noise-reducing optogenetic negative-feedback gene circuits in human 
            cells.
  JOURNAL   Nucleic Acids Res. 2019 Jul 3. pii: 5527975. doi: 
            10.1093/nar/gkz556.
  PUBMED    31269201
REFERENCE   2  (bases 1 to 8036)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8036
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    814..832
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    840..858
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     intron          1041..1613
                     /label=-beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     regulatory      1625..1634
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2771..2827
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2828..3547
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             2828..3544
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2873..2894)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3134..3153)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3481..3502
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3615..3635)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     primer_bind     complement(3988..4005)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3994..4218
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4351..4370)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4502..4549
                     /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             4557..5579
                     /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    5709..5830
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5746..5765)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5800..5819
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5879..5895)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5879..5895)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5892..5914)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5903..5919
                     /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(5927..5957)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5972..5993
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6110..6127)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6281..6869)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6361..6380)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7040..7900)
                     /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     7663..7682
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7901..8005)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcatccctat cagtgataga gatcagatct
      841 ccctatcagt gatagagagc tgttttgacc tccatagaag acaccgggac cgatccagcc
      901 tccggactct agcgtttaaa cttaagcttg gtacccgggg atcctctagg gcctctgagc
      961 tattccagaa gtagtgaaga ggcttttttg gaggcctagg cttttgcaaa aagctccgga
     1021 tcgatcctga gaacttcagg gtgagtttgg ggacccttga ttgttctttc tttttcgcta
     1081 ttgtaaaatt catgttatat ggagggggca aagttttcag ggtgttgttt agaatgggaa
     1141 gatgtccctt gtatcaccat ggaccctcat gataattttg tttctttcac tttctactct
     1201 gttgacaacc attgtctcct cttattttct tttcattttc tgtaactttt tcgttaaact
     1261 ttagcttgca tttgtaacga atttttaaat tcacttttgt ttatttgtca gattgtaagt
     1321 actttctcta atcacttttt tttcaaggca atcagggtat attatattgt acttcagcac
     1381 agttttagag aacaattgtt ataattaaat gataaggtag aatatttctg catataaatt
     1441 ctggctggcg tggaaatatt cttattggta gaaacaacta catcctggtc atcatcctgc
     1501 ctttctcttt atggttacaa tgatatacac tgtttgagat gaggataaaa tactctgagt
     1561 ccaaaccggg cccctctgct aaccatgttc atgccttctt ctttttccta caggtcctgc
     1621 aggcgccacc atgggttcta gactggacaa gagcaaagtc ataaactctg ctctggaatt
     1681 actcaatgaa gtcggtatcg aaggcctgac gacaaggaaa ctcgctcaaa agctgggagt
     1741 tgagcagcct accctgtact ggcacgtgaa gaacaagcgg gccctgctcg atgccctggc
     1801 aatcgagatg ctggacaggc atcataccca cttctgcccc ctggaaggcg agtcatggca
     1861 agactttctg cggaacaacg ccaagtcatt ccgctgtgct ctcctctcac atcgcgacgg
     1921 ggctaaagtg catctcggca cccgcccaac agagaaacag tacgaaaccc tggaaaatca
     1981 gctcgcgttc ctgtgtcagc aaggcttctc cctggagaac gcactgtacg ctctgtccgc
     2041 cgtgggccac tttacactgg gctgcgtatt ggaggatcag gagcatcaag tagcaaaaga
     2101 ggaaagagag acacctacca ccgattctat gcccccactt ctgagacaag caattgagct
     2161 gttcgaccat cagggagccg aacctgcctt ccttttcggc ctggaactaa tcatatgtgg
     2221 cctggagaaa cagctaaagt gcgaaagcgg cgggccaaaa aagaagagaa aggctagcgg
     2281 cgccggcgcc tttttggcta ctacacttga acgtattgag aagaactttg tcattactga
     2341 cccaaggttg ccagataatc ccattatatt cgcgtccgat agtttcttgc agttgacaga
     2401 atatagccgt gaagaaattt tgggaagaaa ctgcaggttt ctacaaggtc ctgaaactga
     2461 tcgcgcgaca gtgagaaaaa ttagagatgc catagataac caaacagagg tcactgttca
     2521 gctgattaat tatacaaaga gtggtaaaaa gttctggaac ctctttcact tgcagcctat
     2581 gcgagatcag aagggagatg tccagtactt tattggggtt cagttggatg gaactgagca
     2641 tgtccgagat gctgccgaga gagagggcgt gatgctggcc aagaagaccg ccgagaacat
     2701 cgacgaggcc gcctggacct ggaacgccta cgccttcgcc gccccctccg gcggcggctc
     2761 cggaagcgga gctactaact tcagcctgct gaagcaggct ggagacgtgg aggagaaccc
     2821 tggacctatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
     2881 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
     2941 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     3001 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca
     3061 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     3121 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     3181 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     3241 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca
     3301 gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     3361 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     3421 caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca
     3481 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     3541 caagtaaacc ggtgcggccg caatcaacct ctggattaca aaatttgtga aagattgact
     3601 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     3661 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     3721 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     3781 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     3841 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     3901 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ctcgagtcta
     3961 gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     4021 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     4081 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     4141 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     4201 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     4261 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     4321 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     4381 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     4441 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtacct
     4501 agaagttcct attccgaagt tcctattctc tagaaagtat aggaacttcc ttggccaaaa
     4561 agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct
     4621 ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag
     4681 ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg
     4741 tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat
     4801 tcagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc
     4861 tgcctgaaac cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg
     4921 ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc
     4981 aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc
     5041 aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc
     5101 tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca
     5161 atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg
     5221 gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg
     5281 agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc
     5341 gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt
     5401 tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga
     5461 ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag
     5521 aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagc
     5581 acgtactacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     5641 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     5701 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     5761 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     5821 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     5881 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     5941 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     6001 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     6061 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     6121 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     6181 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     6241 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     6301 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     6361 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     6421 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     6481 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     6541 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     6601 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     6661 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     6721 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     6781 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     6841 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     6901 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     6961 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     7021 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     7081 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     7141 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     7201 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     7261 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     7321 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     7381 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     7441 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     7501 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     7561 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     7621 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     7681 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     7741 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     7801 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     7861 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     7921 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     7981 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
