LOCUS       Exported                7503 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 7503)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, Jul 1, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7503
                     /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    2465..2599
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2920..2936)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        3410..3514
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3515..4375
                     /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      4546..5134
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    5422..5443
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5458..5488
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5496..5512
                     /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     5520..5536
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    5800..5881
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5998..6396)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(6397..6453)
                     /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(6463..7206)
                     /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(7207..7230)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      7230..7239
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     promoter        complement(7259..7470)
                     /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 gcagactagt agaccacctc ccctgcgagc taagctggac agccaatgac gggtaagaga
     2341 gtgacatttt tcactaacct aagacaggag ggccgtcaga gctactgcct aatccaaaga
     2401 cgggtaaaag tgataaaaat gtatcactcc aacctaagac aggcgcagct tccgagggat
     2461 ttgagatcca gacatgataa gatacattga tgagtttgga caaaccaaaa ctagaatgca
     2521 gtgaaaaaaa tgccttattt gtgaaatttg tgatgctatt gccttatttg taaccattat
     2581 aagctgcaat aaacaagttt gatatctata acaagaaaat atatatataa taagttatca
     2641 cgtaagtaga acatgaaata acaatataat tatcgtatga gttaaatctt aaaagtcacg
     2701 taaaagataa tcatgcgtca ttttgactca cgcggtcgtt atagttcaaa atcagtgaca
     2761 cttaccgcat tgacaagcac gcctcacggg agctccaagc ggcgactgag atgtcctaaa
     2821 tgcacagcga cggattcgcg ctatttagaa agagagagca atatttcaag aatgcatgcg
     2881 tcaattttac gcagactatc tttctagggt taagaattca ctggccgtcg ttttacaacg
     2941 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     3001 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     3061 cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     3121 acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     3181 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     3241 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     3301 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     3361 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     3421 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     3481 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     3541 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     3601 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     3661 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     3721 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     3781 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     3841 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     3901 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     3961 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     4021 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     4081 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     4141 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     4201 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     4261 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     4321 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     4381 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     4441 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     4501 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     4561 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     4621 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     4681 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     4741 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     4801 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     4861 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     4921 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     4981 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     5041 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     5101 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     5161 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     5221 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     5281 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     5341 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     5401 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
     5461 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
     5521 aggaaacagc tatgaccatg attacgccaa ggtcgactta accctagaaa gataatcata
     5581 ttgtgacgta cgttaaagat aatcatgcgt aaaattgacg catgtgtttt atcggtctgt
     5641 atatcgaggt ttatttatta atttgaatag atattaagtt ttattatatt tacacttaca
     5701 tactaataat aaattcaaca aacaatttat ttatgtttat ttatttatta aaaaaaaaca
     5761 aaaactcaaa atttcttcta taaagtaaca aaacttttag cagtgaaaaa aatgctttat
     5821 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     5881 taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt
     5941 ttaaagcaag taaaacctct acaaatgtgg tatggctgat tatgatcctc tggagattta
     6001 gccctcccac acataaccag agggcagcaa ttcacgaatc ccaactgccg tcggctgtcc
     6061 atcactgtcc ttcactatgg ctttgatccc aggatgcaga tcgagaagca cctgtcggca
     6121 ccgtccgcag gggctcaaga tgcccctgtt ctcatttccg atcgcgacga tacaagtcag
     6181 gttgccagct gccgcagcag cagcagtgcc cagcaccacg agttctgcac aaggtccccc
     6241 agtaaaatga tatacattga caccagtgaa gatgcggccg tcgctagaga gagctgcgct
     6301 ggcgacgctg tagtcttcag agatggggat gctgttgatt gtagccgttg ctctttcaat
     6361 gagggtggat tcttcttgag acaaaggctt ggccatgggg ccggggttct cctccacgtc
     6421 gccggcctgc ttcagcaggc tgaagttggt ggcgccgctg cccccgggga gcatgtcaag
     6481 gtcaaaatcg tcaagagcgt cagcaggcag catatcaagg tcaaagtcgt caagggcatc
     6541 ggctgggagc atgtctaagt caaaatcgtc aagggcgtcg gtcggcccgc cgctttcgca
     6601 ctttagctgt ttctccaggc cacatatgat tagttccagg ccgaaaagga aggcaggttc
     6661 ggctccctgc cggtcgaaca gctcaattgc ttgtttcaga agtgggggca tagaatcggt
     6721 ggtaggtgtc tctctttcct cttttgctac ttgatgctcc tgttcctcca atacgcagcc
     6781 cagtgtaaag tggcccacgg cggacagagc gtacagtgcg ttctccaggg agaagccttg
     6841 ctgacacagg aacgcgagct gattttccag ggtttcgtac tgtttctctg ttgggcgggt
     6901 gccgagatgc actttagccc cgtcgcgatg tgagaggaga gcacagcggt atgacttggc
     6961 gttgttccgc agaaagtctt gccatgactc gccttccagg gggcaggagt gggtatgatg
     7021 cctgtccagc atctcgattg gcagggcatc gagcagggcc cgcttgttct tcacgtgcca
     7081 gtacagggta ggctgctcaa ctcccagctt ttgagcgagt ttccttgtcg tcaggccttc
     7141 gataccgact ccattgagta attccagagc agagtttatg actttgctct tgtccagtct
     7201 agacatctta tcgtcatcgt ctttgtaatc catggtggcg gatcccgcgt cacgacacct
     7261 gtgttctggc ggcaaacccg ttgcgaaaaa gaacgttcac ggcgactact gcacttatat
     7321 acggttctcc cccaccctcg ggaaaaaggc ggagccagta cacgacatca ctttcccagt
     7381 ttaccccgcg ccaccttctc taggcaccgg ttcaattgcc gacccctccc cccaacttct
     7441 cggggactgt gggcgatgtg cgctctgccc actgacgggc accggagcca ctcgagtgga
     7501 att
//
