LOCUS       Exported                6090 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 6090)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, Jul 1, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6090
                     /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"
     polyA_signal    1052..1186
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1507..1523)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1997..2101
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2102..2962
                     /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      3133..3721
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    4009..4030
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4045..4075
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4083..4099
                     /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     4107..4123
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    4387..4468
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(4585..4983)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(4984..5040)
                     /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(5050..5793)
                     /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(5794..5817)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      5817..5826
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     promoter        complement(5846..6057)
                     /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 cccgggtaaa actgttttct gcagaaattt actagtgcag
      421 caaccattta aattaaatgt ttgttaacat aatagtgatg gcattttctc ctccccctcc
      481 ttgtggtttt gtccaactag atgttacagt ggcagttgca ctgactgtta agtgtttaaa
      541 tgatgacacc attatgtgaa gtgattttga aatgagagat tccagccaag aattacatct
      601 gctcccatct ccttcaaatc atactctctg gcagtacaga ttatgattga tttgtttgtg
      661 acagattgca ggaaacagtc attgattttt caatatttta ccttaaaatt atttacagtt
      721 gtaaccatgg ggaggtattt tcatgggctg tcagcccctg aaagactagg ataatattcc
      781 ctgctctctg acaagacaaa ttacctgtaa tgagtgcagt agctgaaggg tatactttta
      841 ttttaaaata tgtcaataac cccagtgact aactagtaga ccacctcccc tgcgagctaa
      901 gctggacagc caatgacggg taagagagtg acatttttca ctaacctaag acaggagggc
      961 cgtcagagct actgcctaat ccaaagacgg gtaaaagtga taaaaatgta tcactccaac
     1021 ctaagacagg cgcagcttcc gagggatttg agatccagac atgataagat acattgatga
     1081 gtttggacaa accaaaacta gaatgcagtg aaaaaaatgc cttatttgtg aaatttgtga
     1141 tgctattgcc ttatttgtaa ccattataag ctgcaataaa caagtttgat atctataaca
     1201 agaaaatata tatataataa gttatcacgt aagtagaaca tgaaataaca atataattat
     1261 cgtatgagtt aaatcttaaa agtcacgtaa aagataatca tgcgtcattt tgactcacgc
     1321 ggtcgttata gttcaaaatc agtgacactt accgcattga caagcacgcc tcacgggagc
     1381 tccaagcggc gactgagatg tcctaaatgc acagcgacgg attcgcgcta tttagaaaga
     1441 gagagcaata tttcaagaat gcatgcgtca attttacgca gactatcttt ctagggttaa
     1501 gaattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     1561 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     1621 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt
     1681 tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg
     1741 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     1801 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     1861 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     1921 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     1981 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     2041 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     2101 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     2161 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     2221 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     2281 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     2341 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     2401 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     2461 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     2521 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     2581 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     2641 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     2701 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     2761 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     2821 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     2881 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     2941 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     3001 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     3061 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     3121 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     3181 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     3241 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     3301 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     3361 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     3421 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     3481 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     3541 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     3601 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     3661 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     3721 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     3781 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     3841 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     3901 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     3961 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     4021 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     4081 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaaggt
     4141 cgacttaacc ctagaaagat aatcatattg tgacgtacgt taaagataat catgcgtaaa
     4201 attgacgcat gtgttttatc ggtctgtata tcgaggttta tttattaatt tgaatagata
     4261 ttaagtttta ttatatttac acttacatac taataataaa ttcaacaaac aatttattta
     4321 tgtttattta tttattaaaa aaaaacaaaa actcaaaatt tcttctataa agtaacaaaa
     4381 cttttagcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     4441 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
     4501 ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtat
     4561 ggctgattat gatcctctgg agatttagcc ctcccacaca taaccagagg gcagcaattc
     4621 acgaatccca actgccgtcg gctgtccatc actgtccttc actatggctt tgatcccagg
     4681 atgcagatcg agaagcacct gtcggcaccg tccgcagggg ctcaagatgc ccctgttctc
     4741 atttccgatc gcgacgatac aagtcaggtt gccagctgcc gcagcagcag cagtgcccag
     4801 caccacgagt tctgcacaag gtcccccagt aaaatgatat acattgacac cagtgaagat
     4861 gcggccgtcg ctagagagag ctgcgctggc gacgctgtag tcttcagaga tggggatgct
     4921 gttgattgta gccgttgctc tttcaatgag ggtggattct tcttgagaca aaggcttggc
     4981 catggggccg gggttctcct ccacgtcgcc ggcctgcttc agcaggctga agttggtggc
     5041 gccgctgccc ccggggagca tgtcaaggtc aaaatcgtca agagcgtcag caggcagcat
     5101 atcaaggtca aagtcgtcaa gggcatcggc tgggagcatg tctaagtcaa aatcgtcaag
     5161 ggcgtcggtc ggcccgccgc tttcgcactt tagctgtttc tccaggccac atatgattag
     5221 ttccaggccg aaaaggaagg caggttcggc tccctgccgg tcgaacagct caattgcttg
     5281 tttcagaagt gggggcatag aatcggtggt aggtgtctct ctttcctctt ttgctacttg
     5341 atgctcctgt tcctccaata cgcagcccag tgtaaagtgg cccacggcgg acagagcgta
     5401 cagtgcgttc tccagggaga agccttgctg acacaggaac gcgagctgat tttccagggt
     5461 ttcgtactgt ttctctgttg ggcgggtgcc gagatgcact ttagccccgt cgcgatgtga
     5521 gaggagagca cagcggtatg acttggcgtt gttccgcaga aagtcttgcc atgactcgcc
     5581 ttccaggggg caggagtggg tatgatgcct gtccagcatc tcgattggca gggcatcgag
     5641 cagggcccgc ttgttcttca cgtgccagta cagggtaggc tgctcaactc ccagcttttg
     5701 agcgagtttc cttgtcgtca ggccttcgat accgactcca ttgagtaatt ccagagcaga
     5761 gtttatgact ttgctcttgt ccagtctaga catcttatcg tcatcgtctt tgtaatccat
     5821 ggtggcggat cccgcgtcac gacacctgtg ttctggcggc aaacccgttg cgaaaaagaa
     5881 cgttcacggc gactactgca cttatatacg gttctccccc accctcggga aaaaggcgga
     5941 gccagtacac gacatcactt tcccagttta ccccgcgcca ccttctctag gcaccggttc
     6001 aattgccgac ccctcccccc aacttctcgg ggactgtggg cgatgtgcgc tctgcccact
     6061 gacgggcacc ggagccactc gagtggaatt
//
