LOCUS       sequence-210971-        7275 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Inducible expression of EBFP2 with rtTA for input fluorescence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7275)
  AUTHORS   Davidsohn N, Beal J, Kiani S, Adler A, Yaman F, Li Y, Xie Z, Weiss R
  TITLE     Accurate Predictions of Genetic Circuit Behavior from Part 
            Characterization and Modular Composition.
  JOURNAL   ACS Synth Biol. 2014 Nov 17.
  PUBMED    25369267
REFERENCE   2  (bases 1 to 7275)
  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: "ACS Synth 
            Biol. 2014 Nov 17."
COMMENT     Sequence Label: pExp_TRE:EBFP2
FEATURES             Location/Qualifiers
     source          1..7275
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..21
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    66..84
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    101..119
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    137..155
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    173..191
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    208..226
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    244..262
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        274..312
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     309..333
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    352..376
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      375..384
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(381..383,384..386,387..1100)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     complement(426..447)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    complement(1102..1126)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1216..1235)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1281..1336
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1335..1354)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(1697..1713)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1721..1737
                     /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(1745..1751,1752..1769,1770..1775))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1790..1811
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     misc_feature    1984..2820
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     primer_bind     complement(2850..2870)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2850..2868)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2889..2905)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2913..2929
                     /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(2937..2943,2944..2961,2962..2967))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2982..3003
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3120..3137)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3291..3879)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3371..3390)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4050..4841,4842..4910))
                     /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     4673..4692
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4911..5015)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5041..5496)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5178..5199)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5390..5409
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5623..5645
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5637..5654
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5638..5654
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5664..5683
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5664..5682
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    5697..6500
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     misc_feature    6579..6604
                     /label=SA
                     /note="splice acceptor site"
ORIGIN
        1 caactttgta tagaaaagtt ggctccgaat tcgcccttca ggtccgaggt tctagacgag
       61 tttactccct atcagtgata gagaacgatg tcgagtttac tccctatcag tgatagagaa
      121 cgtatgtcga gtttactccc tatcagtgat agagaacgta tgtcgagttt actccctatc
      181 agtgatagag aacgtatgtc gagtttatcc ctatcagtga tagagaacgt atgtcgagtt
      241 tactccctat cagtgataga gaacgtatgt cgaggtaggc gtgtacggtg ggaggcctat
      301 ataagcagag ctcgtttagt gaaccgtcag atcgcaaagg gcgaattcga cccaagtttg
      361 tacaaaaaag cagcgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc
      421 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg aggggcgagg
      481 gcgagggcga tgccaccaac ggcaagctga ccctgaagtt catctgcacc accggcaagc
      541 tgcccgtgcc ctggcccacc ctcgtgacca ccctgagcca cggcgtgcag tgcttcgccc
      601 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
      661 tccaggagcg caccatcttc ttcaaggacg acggcaccta caagacccgc gccgaggtga
      721 agttcgaggg cgacacccta gtgaaccgca tcgagctgaa gggcgtcgac ttcaaggagg
      781 acggcaacat cctggggcac aagctggagt acaacttcaa cagccacaac atctatatca
      841 tggccgtcaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacgtggagg
      901 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
      961 tgctgctgcc cgacagccac tacctgagca cccagtccgt gctgagcaaa gaccccaacg
     1021 agaagcgcga tcacatggtc ctgctggagt tccgcaccgc cgccgggatc actctcggca
     1081 tggacgagct gtacaagtaa tacccagctt tcttgtacaa agtggtacgc gtgaattcac
     1141 tcctcaggtg caggctgcct atcagaaggt ggtggctggt gtggccaatg ccctggctca
     1201 caaataccac tgagatcttt ttccctctgc caaaaattat ggggacatca tgaagcccct
     1261 tgagcatctg acttctggct aataaaggaa atttattttc attgcaatag tgtgttggaa
     1321 ttttttgtgt ctctcactcg gaaggacata tgggagggca aatcatttaa aacatcagaa
     1381 tgagtatttg gtttagagtt tggcaacata tgcccatatg ctggctgcca tgaacaaagg
     1441 ttggctataa agaggtcatc agtatatgaa acagccccct gctgtccatt ccttattcca
     1501 tagaaaagcc ttgacttgag gttagatttt ttttatattt tgttttgtgt tatttttttc
     1561 tttaacatcc ctaaaatttt ccttacatgt tttactagcc agatttttcc tcctctcctg
     1621 actactccca gtcatagctg tccctcttct cttatggaga tccctcgacc tgcagcccaa
     1681 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     1741 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     1801 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     1861 agcggatcga cagtactaag cttggtctaa tacccaatct ctcgtcttat ccagatgttt
     1921 tatacgcctt aattaatagg gataacaggg taatctcgag gtttaaacgt cgacgcggcc
     1981 gctactaggg acaggattgg tgacagaaaa gccccatcct taggcctcct ccttcctagt
     2041 ctcctgatat tgggtctaac ccccacctcc tgttaggcag attccttatc tggtgacaca
     2101 cccccatttc ctggagccat ctctctcctt gccagaacct ctaaggtttg cttacgatgg
     2161 agccagagag gatcctggga gggagagctt ggcagggggt gggagggaag ggggggatgc
     2221 gtgacctgcc cggttctcag tggccaccct gcgctaccct ctcccagaac ctgagctgct
     2281 ctgacgcggc tgtctggtgc gtttcactga tcctggtgct gcagcttcct tacacttccc
     2341 aagaggagaa gcagtttgga aaaacaaaat cagaataagt tggtcctgag ttctaacttt
     2401 ggctcttcac ctttctagtc cccaatttat attgttcctc cgtgcgtcag ttttacctgt
     2461 gagataaggc cagtagccag ccccgtcctg gcagggctgt ggtgaggagg ggggtgtccg
     2521 tgtggaaaac tccctttgtg agaatggtgc gtcctaggtg ttcaccaggt cgtggccgcc
     2581 tctactccct ttctctttct ccatccttct ttccttaaag agtccccagt gctatctggg
     2641 acatattcct ccgcccagag cagggtcccg cttccctaag gccctgctct gggcttctgg
     2701 gtttgagtcc ttggcaagcc caggagaggc gctcaggctt ccctgtcccc cttcctcgtc
     2761 caccatctca tgcccctggc tctcctgccc cttccctaca ggggttcctg gctctgctct
     2821 ccaccgcggt ggagctccag cttttgttcc ctttagtgag ggttaattgc gcgcttggcg
     2881 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     2941 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     3001 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     3061 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     3121 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     3181 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     3241 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     3301 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     3361 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     3421 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     3481 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     3541 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     3601 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     3661 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     3721 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     3781 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     3841 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     3901 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     3961 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     4021 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     4081 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     4141 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     4201 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     4261 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     4321 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     4381 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     4441 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     4501 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     4561 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     4621 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     4681 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     4741 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     4801 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     4861 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     4921 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     4981 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     5041 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     5101 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     5161 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     5221 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     5281 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     5341 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     5401 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     5461 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc aggctgcgca
     5521 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
     5581 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
     5641 aaacgacggc cagtgagcgc gcgtaatacg actcactata gggcgaattg ggtacctgct
     5701 ttctctgacc agcattctct cccctgggcc tgtgccgctt tctgtctgca gcttgtggcc
     5761 tgggtcacct ctacggctgg cccagatcct tccctgccgc ctccttcagg ttccgtcttc
     5821 ctccactccc tcttcccctt gctctctgct gtgttgctgc ccaaggatgc tctttccgga
     5881 gcacttcctt ctcggcgctg caccacgtga tgtcctctga gcggatcctc cccgtgtctg
     5941 ggtcctctcc gggcatctct cctccctcac ccaaccccat gccgtcttca ctcgctgggt
     6001 tcccttttcc ttctccttct ggggcctgtg ccatctctcg tttcttagga tggccttctc
     6061 cgacggatgt ctcccttgcg tcccgcctcc ccttcttgta ggcctgcatc atcaccgttt
     6121 ttctggacaa ccccaaagta ccccgtctcc ctggctttag ccacctctcc atcctcttgc
     6181 tttctttgcc tggacacccc gttctcctgt ggattcgggt cacctctcac tcctttcatt
     6241 tgggcagctc ccctaccccc cttacctctc tagtctgtgc tagctcttcc agccccctgt
     6301 catggcatct tccaggggtc cgagagctca gctagtcttc ttcctccaac ccgggcccct
     6361 atgtccactt caggacagca tgtttgctgc ctccagggat cctgtgtccc cgagctggga
     6421 ccaccttata ttcccagggc cggttaatgt ggctctggtt ctgggtactt ttatctgtcc
     6481 cctccacccc acagtggggc aagcttatta ccctgttatc cctaaccggt gcgtttttat
     6541 gcttgtagta ttgtataatg tttttaagat ccaagcttct gacctcttct cttcctccca
     6601 cagggcctcg agacaattgc atcgatggta ccagtgaaag tctgcaatga attcgagttg
     6661 gcgcgcctgt cattctaaat ctctctttca gcctaaagct ttttccccgt atccccccag
     6721 gtgtctgcag gctcaaagag cagcgagaag cgttcagagg aaagcgatcc cgtgccacct
     6781 tccccgtgcc cgggctgtcc ccgcacgctg ccggctcggg gatgcggggg gagcgccgga
     6841 ccggagcgga gccccgggcg gctcgctgct gccccctagc gggggaggga cgtaattaca
     6901 tccctggggg ctttgggagg gggctgtccc cgtgagctcc caggcgcgcc tgtcattcta
     6961 aatctctctt tcagcctaaa gctttttccc cgtatccccc caggtgtctg caggctcaaa
     7021 gagcagcgag aagcgttcag aggaaagcga tcccgtgcca ccttccccgt gcccgggctg
     7081 tccccgcacg ctgccggctc ggggatgcgg ggggagcgcc ggaccggagc ggagccccgg
     7141 gcggctcgct gctgccccct agcgggggag ggacgtaatt acatccctgg gggctttggg
     7201 ggggggctgt ccccgtgagc tcccaggcgc gccaactcgg taccgtatcg atgtcgacgt
     7261 taacgctagt gatat
//
