LOCUS       sequence-275842-        7688 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tunable and temporal expression control of NFIL3 and puro resistant 
            gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7688)
  TITLE     Xlone Puro plasmids for inducible gene expression in mammal cells
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7688)
  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: 
            "Unpublished"
COMMENT     Sequence Label: XLone-BSD NFIL3 T2A Puro
FEATURES             Location/Qualifiers
     source          1..7688
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        9..376
                     /label=TRE3GS promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G, modified to eliminate
                     binding sites for endogenous mammalian transcription 
                     factors"
     protein_bind    17..35
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    53..71
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    89..107
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    125..143
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    161..179
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    197..215
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    233..251
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             1790..1843
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1853..2452
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1853..1872)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2349..2369
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    2650..2784
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   3060..3094
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3105..3122)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3105..3121)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3114..3136)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3330..3349)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3449..3471
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3509..3527)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3595..3699
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3700..3768,3769..4560)
                     /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(3918..3937)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4731..5319
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5220..5239
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5473..5490
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5607..5628
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5643..5648,5649..5666,5667..5673)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5681..5697
                     /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     5686..5708
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5705..5721
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5705..5721
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   5747..5809
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5985..6066
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6183..6581)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(6582..6638)
                     /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             complement(6648..7391)
                     /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"
     CDS             complement(7392..7415)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      7415..7424
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(7444..7655)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
ORIGIN
        1 atcacctcga gtttactccc tatcagtgat agagaacgta tgaagagttt actccctatc
       61 agtgatagag aacgtatgca gactttactc cctatcagtg atagagaacg tataaggagt
      121 ttactcccta tcagtgatag agaacgtatg accagtttac tccctatcag tgatagagaa
      181 cgtatctaca gtttactccc tatcagtgat agagaacgta tatccagttt actccctatc
      241 agtgatagag aacgtataag ctttgcttat gtaaaccagg gcgcctataa aagagtgctg
      301 attttttgag taaacttcaa ttccacaaca cttttgtctt ataccaactt tccgtaccac
      361 ttcctaccct cgtaaaggta ccgccaccat gcagctgaga aaaatgcaga ccgtcaaaaa
      421 ggagcaggcg tctcttgatg ccagtagcaa tgtggacaag atgatggtcc ttaattctgc
      481 tttaacggaa gtgtcagaag actccacaac aggtgaggag ctgcttctca gtgaaggaag
      541 tgtggggaag aacaaatctt ctgcatgtcg gaggaaacgg gaattcattc ctgatgaaaa
      601 gaaagatgct atgtattggg aaaaaaggcg gaaaaataat gaagctgcca aaagatctcg
      661 tgagaagcgt cgactgaatg acctggtttt agagaacaaa ctaattgcac tgggagaaga
      721 aaacgccact ttaaaagctg agctgctttc actaaaatta aagtttggtt taattagctc
      781 cacagcatat gctcaagaga ttcagaaact cagtaattct acagctgtgt actttcaaga
      841 ttaccagact tccaaatcca atgtgagttc atttgtggac gagcacgaac cctcgatggt
      901 gtcaagtagt tgtatttctg tcattaaaca ctctccacaa agctcgctgt ccgatgtttc
      961 agaagtgtcc tcagtagaac acacgcagga gagctctgtg cagggaagct gcagaagtcc
     1021 tgaaaacaag ttccagatta tcaagcaaga gccgatggaa ttagagagct acacaaggga
     1081 gccaagagat gaccgaggct cttacacagc gtccatctat caaaactata tggggaattc
     1141 tttctctggg tactcacact ctcccccact actgcaagtc aaccgatcct ccagcaactc
     1201 cccgagaacg tcggaaactg atgatggtgt ggtaggaaag tcatctgatg gagaagacga
     1261 gcaacaggtc cccaagggcc ccatccattc tccagttgaa ctcaagcatg tgcatgcaac
     1321 tgtggttaaa gttccagaag tgaattcctc tgccttgcca cacaagctcc ggatcaaagc
     1381 caaagccatg cagatcaaag tagaagcctt tgataatgaa tttgaggcca cgcaaaaact
     1441 ttcctcacct attgacatga catctaaaag acatttcgaa ctcgaaaagc atagtgcccc
     1501 aagtatggta cattcttctc ttactccttt ctcagtgcaa gtgactaaca ttcaagattg
     1561 gtctctcaaa tcggagcact ggcatcaaaa agaactgagt ggcaaaactc agaatagttt
     1621 caaaactgga gttgttgaaa tgaaagacag tggctacaaa gtttctgacc cagagaactt
     1681 gtatttgaag caggggatag caaacttatc tgcagaggtt gtctcactca agagacttat
     1741 agccacacaa ccaatctctg cttcagactc tggggctagc ggcagcggag agggcagagg
     1801 aagtcttcta acatgcggtg acgtggagga gaatcccggc cctaggctcg agatgaccga
     1861 gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct
     1921 cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat
     1981 cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa
     2041 ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga
     2101 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct
     2161 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg
     2221 gttcctggcc accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt
     2281 cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc
     2341 cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga
     2401 ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gatctagagg
     2461 gcccgtttaa ctagtagacc acctcccctg cgagctaagc tggacagcca atgacgggta
     2521 agagagtgac atttttcact aacctaagac aggagggccg tcagagctac tgcctaatcc
     2581 aaagacgggt aaaagtgata aaaatgtatc actccaacct aagacaggcg cagcttccga
     2641 gggatttgag atccagacat gataagatac attgatgagt ttggacaaac caaaactaga
     2701 atgcagtgaa aaaaatgcct tatttgtgaa atttgtgatg ctattgcctt atttgtaacc
     2761 attataagct gcaataaaca agtttgatat ctataacaag aaaatatata tataataagt
     2821 tatcacgtaa gtagaacatg aaataacaat ataattatcg tatgagttaa atcttaaaag
     2881 tcacgtaaaa gataatcatg cgtcattttg actcacgcgg tcgttatagt tcaaaatcag
     2941 tgacacttac cgcattgaca agcacgcctc acgggagctc caagcggcga ctgagatgtc
     3001 ctaaatgcac agcgacggat tcgcgctatt tagaaagaga gagcaatatt tcaagaatgc
     3061 atgcgtcaat tttacgcaga ctatctttct agggttaaga attcactggc cgtcgtttta
     3121 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     3181 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     3241 cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     3301 atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     3361 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     3421 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     3481 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     3541 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     3601 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     3661 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     3721 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     3781 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     3841 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     3901 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     3961 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     4021 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     4081 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     4141 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     4201 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     4261 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     4321 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     4381 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     4441 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     4501 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     4561 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     4621 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     4681 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     4741 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     4801 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     4861 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     4921 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     4981 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     5041 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     5101 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     5161 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     5221 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     5281 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     5341 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     5401 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     5461 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     5521 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     5581 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     5641 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     5701 cacacaggaa acagctatga ccatgattac gccaaggtcg acttaaccct agaaagataa
     5761 tcatattgtg acgtacgtta aagataatca tgcgtaaaat tgacgcatgt gttttatcgg
     5821 tctgtatatc gaggtttatt tattaatttg aatagatatt aagttttatt atatttacac
     5881 ttacatacta ataataaatt caacaaacaa tttatttatg tttatttatt tattaaaaaa
     5941 aaacaaaaac tcaaaatttc ttctataaag taacaaaact tttagcagtg aaaaaaatgc
     6001 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa
     6061 caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag
     6121 gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga tcctctggag
     6181 atttagccct cccacacata accagagggc agcaattcac gaatcccaac tgccgtcggc
     6241 tgtccatcac tgtccttcac tatggctttg atcccaggat gcagatcgag aagcacctgt
     6301 cggcaccgtc cgcaggggct caagatgccc ctgttctcat ttccgatcgc gacgatacaa
     6361 gtcaggttgc cagctgccgc agcagcagca gtgcccagca ccacgagttc tgcacaaggt
     6421 cccccagtaa aatgatatac attgacacca gtgaagatgc ggccgtcgct agagagagct
     6481 gcgctggcga cgctgtagtc ttcagagatg gggatgctgt tgattgtagc cgttgctctt
     6541 tcaatgaggg tggattcttc ttgagacaaa ggcttggcca tggggccggg gttctcctcc
     6601 acgtcgccgg cctgcttcag caggctgaag ttggtggcgc cgctgccccc ggggagcatg
     6661 tcaaggtcaa aatcgtcaag agcgtcagca ggcagcatat caaggtcaaa gtcgtcaagg
     6721 gcatcggctg ggagcatgtc taagtcaaaa tcgtcaaggg cgtcggtcgg cccgccgctt
     6781 tcgcacttta gctgtttctc caggccacat atgattagtt ccaggccgaa aaggaaggca
     6841 ggttcggctc cctgccggtc gaacagctca attgcttgtt tcagaagtgg gggcatagaa
     6901 tcggtggtag gtgtctctct ttcctctttt gctacttgat gctcctgttc ctccaatacg
     6961 cagcccagtg taaagtggcc cacggcggac agagcgtaca gtgcgttctc cagggagaag
     7021 ccttgctgac acaggaacgc gagctgattt tccagggttt cgtactgttt ctctgttggg
     7081 cgggtgccga gatgcacttt agccccgtcg cgatgtgaga ggagagcaca gcggtatgac
     7141 ttggcgttgt tccgcagaaa gtcttgccat gactcgcctt ccagggggca ggagtgggta
     7201 tgatgcctgt ccagcatctc gattggcagg gcatcgagca gggcccgctt gttcttcacg
     7261 tgccagtaca gggtaggctg ctcaactccc agcttttgag cgagtttcct tgtcgtcagg
     7321 ccttcgatac cgactccatt gagtaattcc agagcagagt ttatgacttt gctcttgtcc
     7381 agtctagaca tcttatcgtc atcgtctttg taatccatgg tggcggatcc cgcgtcacga
     7441 cacctgtgtt ctggcggcaa acccgttgcg aaaaagaacg ttcacggcga ctactgcact
     7501 tatatacggt tctcccccac cctcgggaaa aaggcggagc cagtacacga catcactttc
     7561 ccagtttacc ccgcgccacc ttctctaggc accggttcaa ttgccgaccc ctccccccaa
     7621 cttctcgggg actgtgggcg atgtgcgctc tgcccactga cgggcaccgg agccactcga
     7681 gtggaatt
//
