LOCUS       sequence-450569-        7933 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-one ROSE LP donor construct for TetOn3G-inducible cell line 
            development in HEK293 cell.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7933)
  AUTHORS   Shin SW, Min H, Kim J, Lee JS
  TITLE     A precise and sustainable doxycycline-inducible cell line 
            development platform for reliable mammalian cell engineering with 
            gain-of-function mutations.
  JOURNAL   Metab Eng. 2024 Sep 5;86:12-28. doi: 10.1016/j.ymben.2024.09.004.
  PUBMED    39242074
REFERENCE   2  (bases 1 to 7933)
  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: "Metab 
            Eng."; date: "2024-09-5"; volume: "86:12-28. doi"; pages: " 
            10.1016/j.ymben.2024.09.004"
COMMENT     Sequence Label: EF1-alpha-TetOn3G_2x cHS4_pTRE3G-loxP-EGFP-lox2272
            ...
FEATURES             Location/Qualifiers
     source          1..7933
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        19..123
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(124..192,193..984)
                     /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(342..361)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1155..1743
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1644..1663
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1897..1914
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2283..2784
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     promoter        2913..4075
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3144..4066
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4023..4043
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             4092..4838
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     polyA_signal    4955..5076
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        5652..6030
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    5660..5678
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5696..5714
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5732..5750
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5768..5786
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5804..5822
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5840..5858
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5876..5894
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     5939..5963
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    complement(6040..6073)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      6081..6090
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(6087..6089,6090..6092,6093..6806)
                     /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(6087..6089,6090..6092,6093..6803)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6132..6153)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6393..6412)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6740..6761
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(6813..6846)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     polyA_signal    6963..7084
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    7278..7807
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
ORIGIN
        1 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
       61 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
      121 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
      181 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
      241 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
      301 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
      361 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
      421 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
      481 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
      541 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
      601 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
      661 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
      721 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
      781 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
      841 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
      901 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
      961 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     1021 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     1081 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     1141 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     1201 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     1261 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     1321 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     1381 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     1441 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     1501 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     1561 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     1621 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     1681 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     1741 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     1801 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     1861 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     1921 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     1981 tggcacgaca ggtttcccga ctgagtcggt gtccgtctct ctcctgagtc cggaccactt
     2041 tgagctctac tggcttctgc gccgcctctg gcccactgtt tcccctcccc aggcaggtcc
     2101 tgctttctct gacctgcatt ctctcccctg ggcctgtgcc gctttctgtc tgcagcttgt
     2161 ggcctgggtc acctctacgg ctggcccaga tccttccctg ccgcctcctt caggttccgt
     2221 cttcctccac tccctcttcc ccttgctctc tgctgtgttg ctgcccaagg atgctctttc
     2281 cggagcactt ccttctcggc gctgcaccac gtgatgtcct ctgagcggat cctccccgtg
     2341 tctgggtcct ctccgggcat ctctcctccc tcacccaacc ccatgccgtc ttcactcgct
     2401 gggttccctt ttccttctcc ttctggggcc tgtgccatct ctcgtttctt aggatggcct
     2461 tctccgacgg atgtctccct tgcgtcccgc ctccccttct tgtaggcctg catcatcacc
     2521 gtttttctgg acaaccccaa agtaccccgt ctccctggct ttagccacct ctccatcctc
     2581 ttgctttctt tgcctggaca ccccgttctc ctgtggattc gggtcacctc tcactccttt
     2641 catttgggca gctcccctac cccccttacc tctctagtct gtgctagctc ttccagcccc
     2701 ctgtcatggc atcttccagg ggtccgagag ctcagctagt cttcttcctc caacccgggc
     2761 ccctatgtcc acttcaggac agcatgtttg ctgcctccag ggatcctgtg tccccgagct
     2821 gggaccacct tatattccca gggccggtta atgtggctct ggttctgggt acttttatct
     2881 gtcccctcca ccccacagta taggctttgt gaggctccgg tgcccgtcag tgggcagagc
     2941 gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct
     3001 agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc
     3061 ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca
     3121 acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct
     3181 ttacgggtta tggcccttgc gtgccttgaa ttacttccac ctggctccag tacgtgattc
     3241 ttgatcccga gctggagcca ggggcgggcc ttgcgcttta ggagcccctt cgcctcgtgc
     3301 ttgagttgag gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt ggcaccttcg
     3361 cgcctgtctc gctgctttcg ataagtctct agccatttaa aatttttgat gacctgctgc
     3421 gacgcttttt ttctggcaag atagtcttgt aaatgcgggc caggatctgc acactggtat
     3481 ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca catgttcggc
     3541 gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc aagctggccg
     3601 gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg cggcaaggct
     3661 ggcccggtcg gcaccagttg cgtgagcgga aagatggccg cttcccggcc ctgctccagg
     3721 gggctcaaaa tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac ccacacaaag
     3781 gaaaggggcc tttccgtcct cagccgtcgc ttcatgtgac tccacggagt accgggcgcc
     3841 gtccaggcac ctcgattagt tctggagctt ttggagtacg tcgtctttag gttgggggga
     3901 ggggttttat gcgatggagt ttccccacac tgagtgggtg gagactgaag ttaggccagc
     3961 ttggcacttg atgtaattct ccttggaatt tgcccttttt gagtttggat cttggttcat
     4021 tctcaagcct cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt cgtgaagacg
     4081 tcatcgccac catgtctaga ctggacaaga gcaaagtcat aaactctgct ctggaattac
     4141 tcaatggagt cggtatcgaa ggcctgacga caaggaaact cgctcaaaag ctgggagttg
     4201 agcagcctac cctgtactgg cacgtgaaga acaagcgggc cctgctcgat gccctgccaa
     4261 tcgagatgct ggacaggcat catacccact cctgccccct ggaaggcgag tcatggcaag
     4321 actttctgcg gaacaacgcc aagtcatacc gctgtgctct cctctcacat cgcgacgggg
     4381 ctaaagtgca tctcggcacc cgcccaacag agaaacagta cgaaaccctg gaaaatcagc
     4441 tcgcgttcct gtgtcagcaa ggcttctccc tggagaacgc actgtacgct ctgtccgccg
     4501 tgggccactt tacactgggc tgcgtattgg aggaacagga gcatcaagta gcaaaagagg
     4561 aaagagagac acctaccacc gattctatgc ccccacttct gaaacaagca attgagctgt
     4621 tcgaccggca gggagccgaa cctgccttcc ttttcggcct ggaactaatc atatgtggcc
     4681 tggagaaaca gctaaagtgc gaaagcggcg ggccgaccga cgcccttgac gattttgact
     4741 tagacatgct cccagccgat gcccttgacg actttgacct tgatatgctg cctgctgacg
     4801 ctcttgacga ttttgacctt gacatgctcc ccgggtaaag acgtcattga tcataatcag
     4861 ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa
     4921 cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg
     4981 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     5041 tagttgtggt ttgtccaaac tcatcaatgt atcttaagtg cgatatttaa atggggacag
     5101 ccccccccca aagcccccag ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg
     5161 agccgcccgg ggctccgctc cggtccggcg ctccccccgc atccccgagc cggcagcgtg
     5221 cggggacagc ccgggcacgg ggaaggtggc acgggatcgc tttcctctga acgcttctcg
     5281 ctgctctttg agcctgcaga cacctggggg gatacgggga aaaagcttta ggctgagtgc
     5341 acgatcgata cgtcgctgat atcatttaaa tggggacagc ccccccccaa agcccccagg
     5401 gatgtaatta cgtccctccc ccgctagggg gcagcagcga gccgcccggg gctccgctcc
     5461 ggtccggcgc tccccccgca tccccgagcc ggcagcgtgc ggggacagcc cgggcacggg
     5521 gaaggtggca cgggatcgct ttcctctgaa cgcttctcgc tgctctttga gcctgcagac
     5581 acctgggggg atacggggaa aaagctttag gctgagtgca cgaacgtgga tttcgtcttc
     5641 aagaattcct cgagtttact ccctatcagt gatagagaac gtatgaagag tttactccct
     5701 atcagtgata gagaacgtat gcagacttta ctccctatca gtgatagaga acgtataagg
     5761 agtttactcc ctatcagtga tagagaacgt atgaccagtt tactccctat cagtgataga
     5821 gaacgtatct acagtttact ccctatcagt gatagagaac gtatatccag tttactccct
     5881 atcagtgata gagaacgtat aagctttagg cgtgtacggt gggcgcctat aaaagcagag
     5941 ctcgtttagt gaaccgtcag atcgcctgga gcaattccac aacacttttg tcttatacca
     6001 actttccgta ccacttccta ccctcgtaaa aagcagcgta taacttcgta tagcatacat
     6061 tatacgaagt tatggatccc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg
     6121 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     6181 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     6241 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     6301 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     6361 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     6421 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     6481 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     6541 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     6601 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     6661 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     6721 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     6781 tcggcatgga cgagctgtac aagtaaagat ctataacttc gtataggata ctttatacga
     6841 agttatagac gtcattgatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt
     6901 taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg
     6961 ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     7021 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     7081 cttaagacgt catggggcca ctagggacag gattggtgac agaaaagccc catccttagg
     7141 cctcctcctt cctagtctcc tgatattggg tctaaccccc acctcctgtt aggcagattc
     7201 cttatctggt gacacacccc catttcctgg agccatctct ctccttgcca gaacctctaa
     7261 ggtttgctta cgatggagcc agagaggatc ctgggaggga gagcttggca gggggtggga
     7321 gggaaggggg ggatgcgtga cctgcccggt tctcagtggc caccctgcgc taccctctcc
     7381 cagaacctga gctgctctga cgcggctgtc tggtgcgttt cactgatcct ggtgctgcag
     7441 cttccttaca cttcccaaga ggagaagcag tttggaaaaa caaaatcaga ataagttggt
     7501 cctgagttct aactttggct cttcaccttt ctagtcccca atttatattg ttcctccgtg
     7561 cgtcagtttt acctgtgaga taaggccagt agccagcccc gtcctggcag ggctgtggtg
     7621 aggagggggg tgtccgtgtg gaaaactccc tttgtgagaa tggtgcgtcc taggtgttca
     7681 ccaggtcgtg gccgcctcta ctccctttct ctttctccat ccttctttcc ttaaagagtc
     7741 cccagtgcta tctgggacat attcctccgc ccagagcagg gtcccgcttc cctaaggccc
     7801 tgctctgggc ttctgggttt gagtccttgg caagcccagg agaggcgctc aggcttccct
     7861 gtcccccttc ctcgtccacc atctcatgcc cctggctctc ctgccccttc cctacagggg
     7921 ttcctacttg cgt
//
