LOCUS       Exported                8660 bp ds-DNA     circular SYN 25-JUN-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8660)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, Jul 1, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8660
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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"
     RBS             425..433
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     polyA_signal    3622..3756
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4077..4093)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        4567..4671
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4672..5532
                     /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"
     rep_origin      5703..6291
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    6579..6600
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6615..6645
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6653..6669
                     /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     6677..6693
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    6957..7038
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(7155..7553)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(7554..7610)
                     /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(7620..8363)
                     /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(8364..8387)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      8387..8396
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     promoter        complement(8416..8627)
                     /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 ccggagctac tgtactggct attggaagga ggagattctg
      421 aagataagga ggtaatatta tctcttttaa aagaatactt tcctctgtaa tcctgaatct
      481 ttattacatg taagaacttt gtgcagtaga cagcaatttc tttgaatttg gtatatggaa
      541 acaattttat tttcctctgc taagtttttg agcctgcctc ttctagtgcc atggactgca
      601 ttggtagagc tgagaaatat catttagcca tactcagcac ccttaaaata gcttctttct
      661 gagaattaga tctgtgaagg tgtcctgcac agttcttgta gatgtcattt tagtttgtgg
      721 ttgacgtgca tgcatttagc atgttgctta accgtcctca ttcgcctccc agttctttgt
      781 tgccttcatt tggggggatg tgttttctgc tggatgattt actgctagac atgaccaaac
      841 tctgagtata agacttggtg tttggcagct ggttccagtg ctctgctgag aagtagttgg
      901 gcccagcctg gggcattgta gtgtcctgga ggccgggagc tcctgcacag gggttctttt
      961 cctggttcag agccttaggt gcctttccac ttttcacgta atcttttcct aggttggctt
     1021 gctgcttact ttgcagctgt tgcagggatt cacatggaat ggagggctcc cttttattgt
     1081 ggattatttc tttgataatt tacccatgtg ctttttcatt ttcaaaaacc cagtggtgtt
     1141 taagaatgaa agattcttga agaacaaaaa ttgggttaaa tttgtatcta tcagaaaaga
     1201 ttctatcctc tgatgctatg taggcacatt gaatatgcag gctaaattaa aaacagagta
     1261 aaaccttttt ataatatgcc aattactata tctaagaatg tttatacagg ctgaaatttt
     1321 gtaatggtat ttatatctgt tttccttttt aaggaaaata atatactttt ctaggcatca
     1381 aaaatatctg cccctttcac taatgctgat gtaccacctt cccccaaccc ccaacgctaa
     1441 atttgactgg cttaaaaaca tctgccccct gaactatatc tggggaggaa tattaataaa
     1501 acaatgaagg acttcatggt gtctagttat ataattagga aattggttag aaacgtggct
     1561 aaaaccagtt tctttcatta aaagaactag tgtacattta aaaacaaaaa ccttcaggaa
     1621 aaagactctt tgatctttaa gtcaaatagt atttttggtt aacacagcag gaaggggaaa
     1681 tataccaatt tcagattctt ttatttatgc ctaagtagag gttgtaagca gctaaggaag
     1741 tgattaaaat gtagtctagt aaaaaatggt gctgattact tggaaagcag tttacatttg
     1801 caaaaaaatt cagtattatg accttcacca acttttacac atcatatact tgggttatta
     1861 cttatggtat agcttgtgta tggaatgcaa gggtattttc aaattgacta ggtcttgctg
     1921 gtcatcttaa aatatgttac aatattacaa aattgaaatg taatattttt taatagaagg
     1981 aaaatataaa tttaatatct gggcaattga gacctttaaa cttactttaa aagtatgatc
     2041 ttgatgtata tgatactgtt ttgtctttgc tatattaaca gaattagagg ggtgttctgc
     2101 aattcaaata ccttatatat tccaaatttt attctctata atggactttt aaaataaaag
     2161 gtatatgtgc ttcaagaggg caaaatttga atcatgagct aatttgctaa gcatcagatt
     2221 atagaaaagc atccttgatt aatttggaac tgtgaaaggg ggcgggtaaa actgttttct
     2281 gcagaaattt actagtgcag caaccattta aattaaatgt ttgttaacat aatagtgatg
     2341 gcattttctc ctccccctcc ttgtggtttt gtccaactag atgttacagt ggcagttgca
     2401 ctgactgtta agtgtttaaa tgatgacacc attatgtgaa gtgattttga aatgagagat
     2461 tccagccaag aattacatct gctcccatct ccttcaaatc atactctctg gcagtacaga
     2521 ttatgattga tttgtttgtg acagattgca ggaaacagtc attgattttt caatatttta
     2581 ccttaaaatt atttacagtt gtaaccatgg ggaggtattt tcatgggctg tcagcccctg
     2641 aaagactagg ataatattcc ctgctctctg acaagacaaa ttacctgtaa tgagtgcagt
     2701 agctgaaggg tatactttta ttttaaaata tgtcaataac cccagtgact aaacgaatat
     2761 tgatttagca taatgaagcc tgagtaacgt gaaaatgagc tttttcaagg ggcatggtaa
     2821 agtctttctt tttagctggt tgtaagaagc ttttgattct tttcagccag ctggtaggaa
     2881 tatagaattt tataagcaaa ccatcaggaa tgatagtgtt gtttctgata agcaacatcc
     2941 aaatattttg accctgcttt tagtggtttt tttcaaatct tattttgagt cttactttta
     3001 gtcatagaat agctactgat ttgatgcggt ctttaactga cttaatattt ttacaatttc
     3061 aatatatttt gcattggaat ctccagtaat gaatattaaa atatatgtac aatcatttgt
     3121 agatgatatc aattatatta agacatttca gatgggctat tgtagtattt aatgtgccgt
     3181 attttatggt agaataattc tcagtctctg gacatcaaga ttgctttcag tgggaatgaa
     3241 gattaattta cttcagtcct gattttttag gcatcaatgc atgttttcat ttttgtcaga
     3301 cttttaccct cttttaatgt aattctcaac ttcttatgga tttacttccc aatacataaa
     3361 atccttcaaa acaagaatga taataatttt tatacttttt ataaaaataa atttattttt
     3421 agtccatcaa ggtgtctgaa gactagtaga ccacctcccc tgcgagctaa gctggacagc
     3481 caatgacggg taagagagtg acatttttca ctaacctaag acaggagggc cgtcagagct
     3541 actgcctaat ccaaagacgg gtaaaagtga taaaaatgta tcactccaac ctaagacagg
     3601 cgcagcttcc gagggatttg agatccagac atgataagat acattgatga gtttggacaa
     3661 accaaaacta gaatgcagtg aaaaaaatgc cttatttgtg aaatttgtga tgctattgcc
     3721 ttatttgtaa ccattataag ctgcaataaa caagtttgat atctataaca agaaaatata
     3781 tatataataa gttatcacgt aagtagaaca tgaaataaca atataattat cgtatgagtt
     3841 aaatcttaaa agtcacgtaa aagataatca tgcgtcattt tgactcacgc ggtcgttata
     3901 gttcaaaatc agtgacactt accgcattga caagcacgcc tcacgggagc tccaagcggc
     3961 gactgagatg tcctaaatgc acagcgacgg attcgcgcta tttagaaaga gagagcaata
     4021 tttcaagaat gcatgcgtca attttacgca gactatcttt ctagggttaa gaattcactg
     4081 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     4141 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     4201 tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg
     4261 catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc
     4321 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     4381 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     4441 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     4501 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     4561 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     4621 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     4681 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     4741 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     4801 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     4861 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     4921 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     4981 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     5041 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     5101 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     5161 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     5221 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     5281 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     5341 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     5401 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     5461 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     5521 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     5581 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     5641 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     5701 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     5761 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     5821 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     5881 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     5941 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     6001 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     6061 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     6121 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     6181 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     6241 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     6301 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     6361 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     6421 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     6481 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     6541 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
     6601 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
     6661 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaaggt cgacttaacc
     6721 ctagaaagat aatcatattg tgacgtacgt taaagataat catgcgtaaa attgacgcat
     6781 gtgttttatc ggtctgtata tcgaggttta tttattaatt tgaatagata ttaagtttta
     6841 ttatatttac acttacatac taataataaa ttcaacaaac aatttattta tgtttattta
     6901 tttattaaaa aaaaacaaaa actcaaaatt tcttctataa agtaacaaaa cttttagcag
     6961 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata
     7021 agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg
     7081 gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtat ggctgattat
     7141 gatcctctgg agatttagcc ctcccacaca taaccagagg gcagcaattc acgaatccca
     7201 actgccgtcg gctgtccatc actgtccttc actatggctt tgatcccagg atgcagatcg
     7261 agaagcacct gtcggcaccg tccgcagggg ctcaagatgc ccctgttctc atttccgatc
     7321 gcgacgatac aagtcaggtt gccagctgcc gcagcagcag cagtgcccag caccacgagt
     7381 tctgcacaag gtcccccagt aaaatgatat acattgacac cagtgaagat gcggccgtcg
     7441 ctagagagag ctgcgctggc gacgctgtag tcttcagaga tggggatgct gttgattgta
     7501 gccgttgctc tttcaatgag ggtggattct tcttgagaca aaggcttggc catggggccg
     7561 gggttctcct ccacgtcgcc ggcctgcttc agcaggctga agttggtggc gccgctgccc
     7621 ccggggagca tgtcaaggtc aaaatcgtca agagcgtcag caggcagcat atcaaggtca
     7681 aagtcgtcaa gggcatcggc tgggagcatg tctaagtcaa aatcgtcaag ggcgtcggtc
     7741 ggcccgccgc tttcgcactt tagctgtttc tccaggccac atatgattag ttccaggccg
     7801 aaaaggaagg caggttcggc tccctgccgg tcgaacagct caattgcttg tttcagaagt
     7861 gggggcatag aatcggtggt aggtgtctct ctttcctctt ttgctacttg atgctcctgt
     7921 tcctccaata cgcagcccag tgtaaagtgg cccacggcgg acagagcgta cagtgcgttc
     7981 tccagggaga agccttgctg acacaggaac gcgagctgat tttccagggt ttcgtactgt
     8041 ttctctgttg ggcgggtgcc gagatgcact ttagccccgt cgcgatgtga gaggagagca
     8101 cagcggtatg acttggcgtt gttccgcaga aagtcttgcc atgactcgcc ttccaggggg
     8161 caggagtggg tatgatgcct gtccagcatc tcgattggca gggcatcgag cagggcccgc
     8221 ttgttcttca cgtgccagta cagggtaggc tgctcaactc ccagcttttg agcgagtttc
     8281 cttgtcgtca ggccttcgat accgactcca ttgagtaatt ccagagcaga gtttatgact
     8341 ttgctcttgt ccagtctaga catcttatcg tcatcgtctt tgtaatccat ggtggcggat
     8401 cccgcgtcac gacacctgtg ttctggcggc aaacccgttg cgaaaaagaa cgttcacggc
     8461 gactactgca cttatatacg gttctccccc accctcggga aaaaggcgga gccagtacac
     8521 gacatcactt tcccagttta ccccgcgcca ccttctctag gcaccggttc aattgccgac
     8581 ccctcccccc aacttctcgg ggactgtggg cgatgtgcgc tctgcccact gacgggcacc
     8641 ggagccactc gagtggaatt
//
