LOCUS       Exported                7747 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 7747)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, Jul 1, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7747
                     /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    2709..2843
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3164..3180)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        3654..3758
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3759..4619
                     /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      4790..5378
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    5666..5687
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5702..5732
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5740..5756
                     /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     5764..5780
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    6044..6125
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6242..6640)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(6641..6697)
                     /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(6707..7450)
                     /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(7451..7474)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      7474..7483
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     promoter        complement(7503..7714)
                     /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 ccgaatgttt atacaggctg aaattttgta atggtattta
      421 tatctgtttt cctttttaag gaaaataata tacttttcta ggcatcaaaa atatctgccc
      481 ctttcactaa tgctgatgta ccaccttccc ccaaccccca acgctaaatt tgactggctt
      541 aaaaacatct gccccctgaa ctatatctgg ggaggaatat taataaaaca atgaaggact
      601 tcatggtgtc tagttatata attaggaaat tggttagaaa cgtggctaaa accagtttct
      661 ttcattaaaa gaactagtgt acatttaaaa acaaaaacct tcaggaaaaa gactctttga
      721 tctttaagtc aaatagtatt tttggttaac acagcaggaa ggggaaatat accaatttca
      781 gattctttta tttatgccta agtagaggtt gtaagcagct aaggaagtga ttaaaatgta
      841 gtctagtaaa aaatggtgct gattacttgg aaagcagttt acatttgcaa aaaaattcag
      901 tattatgacc ttcaccaact tttacacatc atatacttgg gttattactt atggtatagc
      961 ttgtgtatgg aatgcaaggg tattttcaaa ttgactaggt cttgctggtc atcttaaaat
     1021 atgttacaat attacaaaat tgaaatgtaa tattttttaa tagaaggaaa atataaattt
     1081 aatatctggg caattgagac ctttaaactt actttaaaag tatgatcttg atgtatatga
     1141 tactgttttg tctttgctat attaacagaa ttagaggggt gttctgcaat tcaaatacct
     1201 tatatattcc aaattttatt ctctataatg gacttttaaa ataaaaggta tatgtgcttc
     1261 aagagggcaa aatttgaatc atgagctaat ttgctaagca tcagattata gaaaagcatc
     1321 cttgattaat ttggaactgt gaaagggggc gggtaaaact gttttctgca gaaatttact
     1381 agtgcagcaa ccatttaaat taaatgtttg ttaacataat agtgatggca ttttctcctc
     1441 cccctccttg tggttttgtc caactagatg ttacagtggc agttgcactg actgttaagt
     1501 gtttaaatga tgacaccatt atgtgaagtg attttgaaat gagagattcc agccaagaat
     1561 tacatctgct cccatctcct tcaaatcata ctctctggca gtacagatta tgattgattt
     1621 gtttgtgaca gattgcagga aacagtcatt gatttttcaa tattttacct taaaattatt
     1681 tacagttgta accatgggga ggtattttca tgggctgtca gcccctgaaa gactaggata
     1741 atattccctg ctctctgaca agacaaatta cctgtaatga gtgcagtagc tgaagggtat
     1801 acttttattt taaaatatgt caataacccc agtgactaaa cgaatattga tttagcataa
     1861 tgaagcctga gtaacgtgaa aatgagcttt ttcaaggggc atggtaaagt ctttcttttt
     1921 agctggttgt aagaagcttt tgattctttt cagccagctg gtaggaatat agaattttat
     1981 aagcaaacca tcaggaatga tagtgttgtt tctgataagc aacatccaaa tattttgacc
     2041 ctgcttttag tggttttttt caaatcttat tttgagtctt acttttagtc atagaatagc
     2101 tactgatttg atgcggtctt taactgactt aatattttta caatttcaat atattttgca
     2161 ttggaatctc cagtaatgaa tattaaaata tatgtacaat catttgtaga tgatatcaat
     2221 tatattaaga catttcagat gggctattgt agtatttaat gtgccgtatt ttatggtaga
     2281 ataattctca gtctctggac atcaagattg ctttcagtgg gaatgaagat taatttactt
     2341 cagtcctgat tttttaggca tcaatgcatg ttttcatttt tgtcagactt ttaccctctt
     2401 ttaatgtaat tctcaacttc ttatggattt acttcccaat acataaaatc cttcaaaaca
     2461 agaatgataa taatttttat actttttata aaaataaatt tatttttagt ccatcaaggt
     2521 gtctgaagac tagtagacca cctcccctgc gagctaagct ggacagccaa tgacgggtaa
     2581 gagagtgaca tttttcacta acctaagaca ggagggccgt cagagctact gcctaatcca
     2641 aagacgggta aaagtgataa aaatgtatca ctccaaccta agacaggcgc agcttccgag
     2701 ggatttgaga tccagacatg ataagataca ttgatgagtt tggacaaacc aaaactagaa
     2761 tgcagtgaaa aaaatgcctt atttgtgaaa tttgtgatgc tattgcctta tttgtaacca
     2821 ttataagctg caataaacaa gtttgatatc tataacaaga aaatatatat ataataagtt
     2881 atcacgtaag tagaacatga aataacaata taattatcgt atgagttaaa tcttaaaagt
     2941 cacgtaaaag ataatcatgc gtcattttga ctcacgcggt cgttatagtt caaaatcagt
     3001 gacacttacc gcattgacaa gcacgcctca cgggagctcc aagcggcgac tgagatgtcc
     3061 taaatgcaca gcgacggatt cgcgctattt agaaagagag agcaatattt caagaatgca
     3121 tgcgtcaatt ttacgcagac tatctttcta gggttaagaa ttcactggcc gtcgttttac
     3181 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
     3241 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
     3301 gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     3361 tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     3421 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     3481 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     3541 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     3601 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     3661 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     3721 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     3781 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     3841 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     3901 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     3961 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     4021 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     4081 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     4141 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     4201 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     4261 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     4321 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     4381 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     4441 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     4501 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     4561 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     4621 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     4681 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     4741 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     4801 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     4861 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     4921 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     4981 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     5041 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     5101 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     5161 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     5221 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     5281 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     5341 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     5401 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     5461 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     5521 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     5581 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     5641 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
     5701 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
     5761 acacaggaaa cagctatgac catgattacg ccaaggtcga cttaacccta gaaagataat
     5821 catattgtga cgtacgttaa agataatcat gcgtaaaatt gacgcatgtg ttttatcggt
     5881 ctgtatatcg aggtttattt attaatttga atagatatta agttttatta tatttacact
     5941 tacatactaa taataaattc aacaaacaat ttatttatgt ttatttattt attaaaaaaa
     6001 aacaaaaact caaaatttct tctataaagt aacaaaactt ttagcagtga aaaaaatgct
     6061 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
     6121 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg
     6181 ttttttaaag caagtaaaac ctctacaaat gtggtatggc tgattatgat cctctggaga
     6241 tttagccctc ccacacataa ccagagggca gcaattcacg aatcccaact gccgtcggct
     6301 gtccatcact gtccttcact atggctttga tcccaggatg cagatcgaga agcacctgtc
     6361 ggcaccgtcc gcaggggctc aagatgcccc tgttctcatt tccgatcgcg acgatacaag
     6421 tcaggttgcc agctgccgca gcagcagcag tgcccagcac cacgagttct gcacaaggtc
     6481 ccccagtaaa atgatataca ttgacaccag tgaagatgcg gccgtcgcta gagagagctg
     6541 cgctggcgac gctgtagtct tcagagatgg ggatgctgtt gattgtagcc gttgctcttt
     6601 caatgagggt ggattcttct tgagacaaag gcttggccat ggggccgggg ttctcctcca
     6661 cgtcgccggc ctgcttcagc aggctgaagt tggtggcgcc gctgcccccg gggagcatgt
     6721 caaggtcaaa atcgtcaaga gcgtcagcag gcagcatatc aaggtcaaag tcgtcaaggg
     6781 catcggctgg gagcatgtct aagtcaaaat cgtcaagggc gtcggtcggc ccgccgcttt
     6841 cgcactttag ctgtttctcc aggccacata tgattagttc caggccgaaa aggaaggcag
     6901 gttcggctcc ctgccggtcg aacagctcaa ttgcttgttt cagaagtggg ggcatagaat
     6961 cggtggtagg tgtctctctt tcctcttttg ctacttgatg ctcctgttcc tccaatacgc
     7021 agcccagtgt aaagtggccc acggcggaca gagcgtacag tgcgttctcc agggagaagc
     7081 cttgctgaca caggaacgcg agctgatttt ccagggtttc gtactgtttc tctgttgggc
     7141 gggtgccgag atgcacttta gccccgtcgc gatgtgagag gagagcacag cggtatgact
     7201 tggcgttgtt ccgcagaaag tcttgccatg actcgccttc cagggggcag gagtgggtat
     7261 gatgcctgtc cagcatctcg attggcaggg catcgagcag ggcccgcttg ttcttcacgt
     7321 gccagtacag ggtaggctgc tcaactccca gcttttgagc gagtttcctt gtcgtcaggc
     7381 cttcgatacc gactccattg agtaattcca gagcagagtt tatgactttg ctcttgtcca
     7441 gtctagacat cttatcgtca tcgtctttgt aatccatggt ggcggatccc gcgtcacgac
     7501 acctgtgttc tggcggcaaa cccgttgcga aaaagaacgt tcacggcgac tactgcactt
     7561 atatacggtt ctcccccacc ctcgggaaaa aggcggagcc agtacacgac atcactttcc
     7621 cagtttaccc cgcgccacct tctctaggca ccggttcaat tgccgacccc tccccccaac
     7681 ttctcgggga ctgtgggcga tgtgcgctct gcccactgac gggcaccgga gccactcgag
     7741 tggaatt
//
