LOCUS       sequence-358578-        6702 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  1xTRE Tet-On cassette with 3xp3 GFP marker for generating knockins 
            with doxycycline-inducible rescue..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6702)
  AUTHORS   Chen J, Luo J, Gurav AS, Chen Z, Wang Y, Montell C
  TITLE     A DREaMR system to simplify combining mutations with rescue 
            transgenes in Aedes aegypti.
  JOURNAL   Genetics. 2021 Nov 5;219(3). pii: 6368066. doi: 
            10.1093/genetics/iyab146.
  PUBMED    34740249
REFERENCE   2  (bases 1 to 6702)
  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: "Genetics.";
            date: "2021-11-5"; volume: "219(3). pii: 6368066. doi"; pages: " 
            10.1093/genetics/iyab146"
COMMENT     Sequence Label: 1xTRE DREaMR 3p3GFP
FEATURES             Location/Qualifiers
     source          1..6702
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        24..128
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(129..197,198..989)
                     /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     complement(347..366)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1160..1748
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1649..1668
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1902..1919
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2036..2057
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2072..2077,2078..2095,2096..2102)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2110..2126
                     /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)."
     primer_bind     2115..2137
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2134..2150
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2134..2150
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   2641..2675
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    3726..3860
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     regulatory      4121..4130
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4127..4129,4130..4132,4133..4846)
                     /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             join(4127..4129,4130..4132,4133..4843)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4172..4193)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4433..4452)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4780..4801
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4987..5108
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    5147..5165
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5183..5201
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5218..5236
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5254..5272
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5290..5308
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5325..5343
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5361..5379
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             5564..5653
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     repeat_region   5895..5957
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6087..6104)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6087..6103)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6096..6118)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6245..6700
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6332..6351)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6542..6563
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
       61 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
      121 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
      181 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
      241 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
      301 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
      361 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
      421 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
      481 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
      541 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
      601 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
      661 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
      721 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
      781 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
      841 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
      901 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
      961 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     1021 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     1081 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     1141 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     1201 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     1261 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     1321 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     1381 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     1441 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     1501 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     1561 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     1621 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     1681 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     1741 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     1801 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     1861 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     1921 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     1981 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     2041 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     2101 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     2161 ccaagcgcgc ccgccgggta actcacgggg tatccatgtc catttctgcg gcatccagcc
     2221 aggatacccg tcctcgctga cgtaatatcc cagcgccgca ccgctgtcat taatctgcac
     2281 accggcacgg cagttccggc tgtcgccggt attgttcggg ttgctgatgc gcttcgggct
     2341 gaccatccgg aactgtgtcc ggaaaagccg cgacgaactg gtatcccagg tggcctgaac
     2401 gaacagttca ccgttaaagg cgtgcatggc cacaccttcc cgaatcatca tggtaaacgt
     2461 gcgttttcgc tcaacgtcaa tgcagcagca gtcatcctcg gcaaactctt tccatgccgc
     2521 ttcaacctcg cgggaaaagg cacgggcttc ttcctccccg atgcccagat agcgccagct
     2581 tgggcgatga ctgagccgga aaaaagaccc gacgatatga tcctgatgca gctagattaa
     2641 ccctagaaag atagtctgcg taaaattgac gcatgcattc ttgaaatatt gctctctctt
     2701 tctaaatagc gcgaatccgt cgctgtgcat ttaggacatc tcagtcgccg cttggagctc
     2761 ccgtgaggcg tgcttgtcaa tgcggtaagt gtcactgatt ttgaactata acgaccgcgt
     2821 gagtcaaaat gacgcatgat tatcttttac gtgactttta agatttaact catacgataa
     2881 ttatattgtt atttcatgtt ctacttacgt gataacttat tatatatata ttttcttgtt
     2941 atagatatca agctacgtag ctagccacca tgagtaggtt ggacaagtcc aaggttatca
     3001 acggcgcttt ggagttgttg aacggtgttg gcattgaggg tctgaccact cgcaaactgg
     3061 cccaaaagct cggagtagaa cagcccacct tgtattggca tgtgaaaaac aagagggctt
     3121 tgttggacgc tctgccgata gagatgttgg accgtcatca cactcatttt tgccccctgg
     3181 aaggagagtc ctggcaggat ttcctccgta acaatgccaa aagttttcgg tgcgcactcc
     3241 tgagtcatcg agacggcgct aaagtacacc tgggaacccg gccaactgag aagcagtatg
     3301 agacactgga gaaccaactg gcattcctgt gtcaacaagg cttcagtttg gaaaatgccc
     3361 tgtacgcctt gagcgcggtt ggacacttta cactgggttg cgttttggag gaacaggaac
     3421 atcaggtcgc gaaggaggaa agggagacac cgaccacaga ttcgatgccg cctctgctcc
     3481 gtcaagcaat tgagctcttt gaccgacaag gagcagaacc tgcctttctc tttggtctcg
     3541 aactcatcat ttgtggattg gaaaagcagc tcaaatgtga gtcgggagga cctgccgacg
     3601 cgctggatga ctttgatttg gacatgctcc cggcagacgc cctggatgat ttcgatctcg
     3661 atatgctgcc cgcagacgct ttggatgact ttgacctgga tatgttgccg ggataactaa
     3721 gtaaggatcc agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc
     3781 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     3841 taagctgcaa taaacaagtt gcggccgctc tagaactagt gccctttcgt cttcaagaat
     3901 tggatctaat tcaattagag actaattcaa ttagagctaa ttcaattagg atccaagctt
     3961 atcgatttcg aaccctcgac cgccggagta taaatagagg cgcttcgtct acggagcgac
     4021 aattcaattc aaacaagcaa agtgaacacg tcgctaagcg aaagctaagc aaataaacaa
     4081 gcgcagctga acaagctaaa caatcggctc gaagccggtc gccaccatgg tgagcaaggg
     4141 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     4201 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     4261 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     4321 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     4381 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     4441 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     4501 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     4561 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
     4621 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     4681 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     4741 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     4801 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaaagcg gcctaagcga
     4861 tcgctcaggc cgcgactcta gatcataatc agccatacca catttgtaga ggttttactt
     4921 gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     4981 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     5041 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     5101 gtatcttaac gcgagttaag cttatcgata ccgtcgacga gtttactccc tatcagtgat
     5161 agagaacgta tgtcgagttt actccctatc agtgatagag aacgatgtcg agtttactcc
     5221 ctatcagtga tagagaacgt atgtcgagtt tactccctat cagtgataga gaacgtatgt
     5281 cgagtttact ccctatcagt gatagagaac gtatgtcgag tttatcccta tcagtgatag
     5341 agaacgtatg tcgagtttac tccctatcag tgatagagaa cgtatgtcga ggagctcgcc
     5401 cggggatcga gcgcagcggt ataaaagggc gcggggtggc tgagagcatc agttgtgaat
     5461 gaatgttcga gccgagcaga cgtgccgctg ccttcgttaa tatcctttga ataagccaac
     5521 tttgaatcac aagacgcata ccaaacggta ccgcggcacc atgtacccct acgacgtgcc
     5581 cgactacgcc ggctacccat acgacgtccc agattacgct ggttcgtacc cgtacgacgt
     5641 gccagattac gcctccggcg gttccggtgg cagcggcggc gccgttaatt aagaggatcc
     5701 cccgggctgc aggaattcga taaaagtttt gttactttat agaagaaatt ttgagttttt
     5761 gtttttttta ataaataaat aaacataaat aaattgtttg ttgaatttat tattagtatg
     5821 taagtgtaaa tataataaaa cttaatatct attcaaatta ataaataaac ctcgatatac
     5881 agaccgataa aacacatgcg tcaattttac acatgattat ctttaacgta cgtcacaata
     5941 tgattatctt tctagggtta atctagctgc gtgttctgca gcgtgtcgag catcttcatc
     6001 tgctccatca cgctgtaaaa cacatttgca ccgcgagtct gcccgtcctc cacgggttca
     6061 aaaacgtgaa tgaacgaggc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa
     6121 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     6181 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     6241 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     6301 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     6361 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     6421 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     6481 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     6541 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     6601 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     6661 tttaacgcga attttaacaa aatattaacg cttacaattt ag
//
