LOCUS       Exported                6533 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 6533)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, Jul 1, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6533
                     /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"
     misc_feature    657..659
                     /label=PAM mutation
     RBS             965..973
                     /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    1495..1629
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1950..1966)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2440..2544
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2545..3405
                     /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      3576..4164
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    4452..4473
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4488..4518
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4526..4542
                     /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     4550..4566
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    4830..4911
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5028..5426)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(5427..5483)
                     /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(5493..6236)
                     /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(6237..6260)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6260..6269
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     promoter        complement(6289..6500)
                     /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 ccgccgcagc cgcagcccct ggcgctaacg gtcggtaacg
      421 gcccgcgcgc gccgcccgcc gggggctcgc gccagccacg agggagcgtc cgcggcccgc
      481 gcgcccgcgc ggcggaggag aggtgagccc ccgcccgggc caggccctct ggccgcgccg
      541 tccgcccctc tagtcgtgtc ccctcgtggg ccgaacggac gcggcggtgc cccgcgcccg
      601 accagacgtc ccgtgggcta gggcctgggc ctcgggccgc gtcggcgccg gtcgaggttc
      661 tccgggtgtc ggggttcggg gcgggcgcgc gtgggcgtgg ctcctctgtc cacgcctgtt
      721 cccttcgtcg ccgcggctct cgtccgggac acggctttcc ggagtagagc ccttggagta
      781 gagcccttgg aggtgttaag tgtgatgctt ccataataca tttggatgct gtcagctaag
      841 ttcacttctg aactaagggg ttcctccaaa tgttggctga aattcatccc aaggctggtc
      901 tgcaaagtct gcaattcata atggagctac tgtactggct attggaagga ggagattctg
      961 aagataagga ggtaaaacct gtttagaaat taaaaatgag ttacgattta aagaaaattc
     1021 agatgactca ttgtgagtgc tagttctctt gtaggatgcc actggaaatg ttgaaatgaa
     1081 aaatattcag ccgttggtct ttgaaatttc ctgtgatgtg tttcaatcta gatgcaaaga
     1141 acatggaaaa atcaaagtgc tcgagtggtt taaatatgtt ttgggtattc ctgtttatag
     1201 actataatac ttttccaatt aaaatcctca gttgtcacgc agaagaaggt taagctgtat
     1261 ttgattgcca gttttactga aaatgcttag tattttacag tatcaccaaa tataactagt
     1321 agaccacctc ccctgcgagc taagctggac agccaatgac gggtaagaga gtgacatttt
     1381 tcactaacct aagacaggag ggccgtcaga gctactgcct aatccaaaga cgggtaaaag
     1441 tgataaaaat gtatcactcc aacctaagac aggcgcagct tccgagggat ttgagatcca
     1501 gacatgataa gatacattga tgagtttgga caaaccaaaa ctagaatgca gtgaaaaaaa
     1561 tgccttattt gtgaaatttg tgatgctatt gccttatttg taaccattat aagctgcaat
     1621 aaacaagttt gatatctata acaagaaaat atatatataa taagttatca cgtaagtaga
     1681 acatgaaata acaatataat tatcgtatga gttaaatctt aaaagtcacg taaaagataa
     1741 tcatgcgtca ttttgactca cgcggtcgtt atagttcaaa atcagtgaca cttaccgcat
     1801 tgacaagcac gcctcacggg agctccaagc ggcgactgag atgtcctaaa tgcacagcga
     1861 cggattcgcg ctatttagaa agagagagca atatttcaag aatgcatgcg tcaattttac
     1921 gcagactatc tttctagggt taagaattca ctggccgtcg ttttacaacg tcgtgactgg
     1981 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     2041 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     2101 gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     2161 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     2221 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     2281 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     2341 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     2401 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     2461 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     2521 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     2581 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     2641 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     2701 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     2761 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     2821 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     2881 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     2941 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     3001 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     3061 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     3121 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     3181 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     3241 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     3301 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     3361 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     3421 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     3481 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     3541 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     3601 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     3661 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     3721 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     3781 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     3841 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     3901 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     3961 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     4021 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     4081 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     4141 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     4201 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     4261 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     4321 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
     4381 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     4441 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
     4501 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     4561 tatgaccatg attacgccaa ggtcgactta accctagaaa gataatcata ttgtgacgta
     4621 cgttaaagat aatcatgcgt aaaattgacg catgtgtttt atcggtctgt atatcgaggt
     4681 ttatttatta atttgaatag atattaagtt ttattatatt tacacttaca tactaataat
     4741 aaattcaaca aacaatttat ttatgtttat ttatttatta aaaaaaaaca aaaactcaaa
     4801 atttcttcta taaagtaaca aaacttttag cagtgaaaaa aatgctttat ttgtgaaatt
     4861 tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac
     4921 aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag
     4981 taaaacctct acaaatgtgg tatggctgat tatgatcctc tggagattta gccctcccac
     5041 acataaccag agggcagcaa ttcacgaatc ccaactgccg tcggctgtcc atcactgtcc
     5101 ttcactatgg ctttgatccc aggatgcaga tcgagaagca cctgtcggca ccgtccgcag
     5161 gggctcaaga tgcccctgtt ctcatttccg atcgcgacga tacaagtcag gttgccagct
     5221 gccgcagcag cagcagtgcc cagcaccacg agttctgcac aaggtccccc agtaaaatga
     5281 tatacattga caccagtgaa gatgcggccg tcgctagaga gagctgcgct ggcgacgctg
     5341 tagtcttcag agatggggat gctgttgatt gtagccgttg ctctttcaat gagggtggat
     5401 tcttcttgag acaaaggctt ggccatgggg ccggggttct cctccacgtc gccggcctgc
     5461 ttcagcaggc tgaagttggt ggcgccgctg cccccgggga gcatgtcaag gtcaaaatcg
     5521 tcaagagcgt cagcaggcag catatcaagg tcaaagtcgt caagggcatc ggctgggagc
     5581 atgtctaagt caaaatcgtc aagggcgtcg gtcggcccgc cgctttcgca ctttagctgt
     5641 ttctccaggc cacatatgat tagttccagg ccgaaaagga aggcaggttc ggctccctgc
     5701 cggtcgaaca gctcaattgc ttgtttcaga agtgggggca tagaatcggt ggtaggtgtc
     5761 tctctttcct cttttgctac ttgatgctcc tgttcctcca atacgcagcc cagtgtaaag
     5821 tggcccacgg cggacagagc gtacagtgcg ttctccaggg agaagccttg ctgacacagg
     5881 aacgcgagct gattttccag ggtttcgtac tgtttctctg ttgggcgggt gccgagatgc
     5941 actttagccc cgtcgcgatg tgagaggaga gcacagcggt atgacttggc gttgttccgc
     6001 agaaagtctt gccatgactc gccttccagg gggcaggagt gggtatgatg cctgtccagc
     6061 atctcgattg gcagggcatc gagcagggcc cgcttgttct tcacgtgcca gtacagggta
     6121 ggctgctcaa ctcccagctt ttgagcgagt ttccttgtcg tcaggccttc gataccgact
     6181 ccattgagta attccagagc agagtttatg actttgctct tgtccagtct agacatctta
     6241 tcgtcatcgt ctttgtaatc catggtggcg gatcccgcgt cacgacacct gtgttctggc
     6301 ggcaaacccg ttgcgaaaaa gaacgttcac ggcgactact gcacttatat acggttctcc
     6361 cccaccctcg ggaaaaaggc ggagccagta cacgacatca ctttcccagt ttaccccgcg
     6421 ccaccttctc taggcaccgg ttcaattgcc gacccctccc cccaacttct cggggactgt
     6481 gggcgatgtg cgctctgccc actgacgggc accggagcca ctcgagtgga att
//
