LOCUS       Exported                8590 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 8590)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, Jul 1, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8590
                     /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    3552..3686
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4007..4023)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        4497..4601
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4602..5462
                     /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      5633..6221
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    6509..6530
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6545..6575
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6583..6599
                     /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     6607..6623
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    6887..6968
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(7085..7483)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(7484..7540)
                     /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(7550..8293)
                     /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(8294..8317)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      8317..8326
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     promoter        complement(8346..8557)
                     /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 aggcaattga gacctttaaa cttactttaa aagtatgatc ttgatgtata
     1981 tgatactgtt ttgtctttgc tatattaaca gaattagagg ggtgttctgc aattcaaata
     2041 ccttatatat tccaaatttt attctctata atggactttt aaaataaaag gtatatgtgc
     2101 ttcaagaggg caaaatttga atcatgagct aatttgctaa gcatcagatt atagaaaagc
     2161 atccttgatt aatttggaac tgtgaaaggg ggcgggtaaa actgttttct gcagaaattt
     2221 actagtgcag caaccattta aattaaatgt ttgttaacat aatagtgatg gcattttctc
     2281 ctccccctcc ttgtggtttt gtccaactag atgttacagt ggcagttgca ctgactgtta
     2341 agtgtttaaa tgatgacacc attatgtgaa gtgattttga aatgagagat tccagccaag
     2401 aattacatct gctcccatct ccttcaaatc atactctctg gcagtacaga ttatgattga
     2461 tttgtttgtg acagattgca ggaaacagtc attgattttt caatatttta ccttaaaatt
     2521 atttacagtt gtaaccatgg ggaggtattt tcatgggctg tcagcccctg aaagactagg
     2581 ataatattcc ctgctctctg acaagacaaa ttacctgtaa tgagtgcagt agctgaaggg
     2641 tatactttta ttttaaaata tgtcaataac cccagtgact aaacgaatat tgatttagca
     2701 taatgaagcc tgagtaacgt gaaaatgagc tttttcaagg ggcatggtaa agtctttctt
     2761 tttagctggt tgtaagaagc ttttgattct tttcagccag ctggtaggaa tatagaattt
     2821 tataagcaaa ccatcaggaa tgatagtgtt gtttctgata agcaacatcc aaatattttg
     2881 accctgcttt tagtggtttt tttcaaatct tattttgagt cttactttta gtcatagaat
     2941 agctactgat ttgatgcggt ctttaactga cttaatattt ttacaatttc aatatatttt
     3001 gcattggaat ctccagtaat gaatattaaa atatatgtac aatcatttgt agatgatatc
     3061 aattatatta agacatttca gatgggctat tgtagtattt aatgtgccgt attttatggt
     3121 agaataattc tcagtctctg gacatcaaga ttgctttcag tgggaatgaa gattaattta
     3181 cttcagtcct gattttttag gcatcaatgc atgttttcat ttttgtcaga cttttaccct
     3241 cttttaatgt aattctcaac ttcttatgga tttacttccc aatacataaa atccttcaaa
     3301 acaagaatga taataatttt tatacttttt ataaaaataa atttattttt agtccatcaa
     3361 ggtgtctgaa gactagtaga ccacctcccc tgcgagctaa gctggacagc caatgacggg
     3421 taagagagtg acatttttca ctaacctaag acaggagggc cgtcagagct actgcctaat
     3481 ccaaagacgg gtaaaagtga taaaaatgta tcactccaac ctaagacagg cgcagcttcc
     3541 gagggatttg agatccagac atgataagat acattgatga gtttggacaa accaaaacta
     3601 gaatgcagtg aaaaaaatgc cttatttgtg aaatttgtga tgctattgcc ttatttgtaa
     3661 ccattataag ctgcaataaa caagtttgat atctataaca agaaaatata tatataataa
     3721 gttatcacgt aagtagaaca tgaaataaca atataattat cgtatgagtt aaatcttaaa
     3781 agtcacgtaa aagataatca tgcgtcattt tgactcacgc ggtcgttata gttcaaaatc
     3841 agtgacactt accgcattga caagcacgcc tcacgggagc tccaagcggc gactgagatg
     3901 tcctaaatgc acagcgacgg attcgcgcta tttagaaaga gagagcaata tttcaagaat
     3961 gcatgcgtca attttacgca gactatcttt ctagggttaa gaattcactg gccgtcgttt
     4021 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     4081 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     4141 tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg
     4201 gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     4261 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     4321 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     4381 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta
     4441 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     4501 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     4561 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     4621 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     4681 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     4741 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     4801 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     4861 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     4921 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     4981 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     5041 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     5101 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     5161 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     5221 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     5281 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     5341 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     5401 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     5461 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     5521 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     5581 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     5641 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     5701 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     5761 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     5821 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     5881 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     5941 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     6001 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     6061 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     6121 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     6181 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     6241 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     6301 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     6361 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
     6421 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     6481 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
     6541 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
     6601 ttcacacagg aaacagctat gaccatgatt acgccaaggt cgacttaacc ctagaaagat
     6661 aatcatattg tgacgtacgt taaagataat catgcgtaaa attgacgcat gtgttttatc
     6721 ggtctgtata tcgaggttta tttattaatt tgaatagata ttaagtttta ttatatttac
     6781 acttacatac taataataaa ttcaacaaac aatttattta tgtttattta tttattaaaa
     6841 aaaaacaaaa actcaaaatt tcttctataa agtaacaaaa cttttagcag tgaaaaaaat
     6901 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
     6961 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg
     7021 aggtttttta aagcaagtaa aacctctaca aatgtggtat ggctgattat gatcctctgg
     7081 agatttagcc ctcccacaca taaccagagg gcagcaattc acgaatccca actgccgtcg
     7141 gctgtccatc actgtccttc actatggctt tgatcccagg atgcagatcg agaagcacct
     7201 gtcggcaccg tccgcagggg ctcaagatgc ccctgttctc atttccgatc gcgacgatac
     7261 aagtcaggtt gccagctgcc gcagcagcag cagtgcccag caccacgagt tctgcacaag
     7321 gtcccccagt aaaatgatat acattgacac cagtgaagat gcggccgtcg ctagagagag
     7381 ctgcgctggc gacgctgtag tcttcagaga tggggatgct gttgattgta gccgttgctc
     7441 tttcaatgag ggtggattct tcttgagaca aaggcttggc catggggccg gggttctcct
     7501 ccacgtcgcc ggcctgcttc agcaggctga agttggtggc gccgctgccc ccggggagca
     7561 tgtcaaggtc aaaatcgtca agagcgtcag caggcagcat atcaaggtca aagtcgtcaa
     7621 gggcatcggc tgggagcatg tctaagtcaa aatcgtcaag ggcgtcggtc ggcccgccgc
     7681 tttcgcactt tagctgtttc tccaggccac atatgattag ttccaggccg aaaaggaagg
     7741 caggttcggc tccctgccgg tcgaacagct caattgcttg tttcagaagt gggggcatag
     7801 aatcggtggt aggtgtctct ctttcctctt ttgctacttg atgctcctgt tcctccaata
     7861 cgcagcccag tgtaaagtgg cccacggcgg acagagcgta cagtgcgttc tccagggaga
     7921 agccttgctg acacaggaac gcgagctgat tttccagggt ttcgtactgt ttctctgttg
     7981 ggcgggtgcc gagatgcact ttagccccgt cgcgatgtga gaggagagca cagcggtatg
     8041 acttggcgtt gttccgcaga aagtcttgcc atgactcgcc ttccaggggg caggagtggg
     8101 tatgatgcct gtccagcatc tcgattggca gggcatcgag cagggcccgc ttgttcttca
     8161 cgtgccagta cagggtaggc tgctcaactc ccagcttttg agcgagtttc cttgtcgtca
     8221 ggccttcgat accgactcca ttgagtaatt ccagagcaga gtttatgact ttgctcttgt
     8281 ccagtctaga catcttatcg tcatcgtctt tgtaatccat ggtggcggat cccgcgtcac
     8341 gacacctgtg ttctggcggc aaacccgttg cgaaaaagaa cgttcacggc gactactgca
     8401 cttatatacg gttctccccc accctcggga aaaaggcgga gccagtacac gacatcactt
     8461 tcccagttta ccccgcgcca ccttctctag gcaccggttc aattgccgac ccctcccccc
     8521 aacttctcgg ggactgtggg cgatgtgcgc tctgcccact gacgggcacc ggagccactc
     8581 gagtggaatt
//
