LOCUS       Exported                7142 bp ds-DNA     circular SYN 17-OCT-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7142)
  AUTHORS   giselle
  TITLE     Direct Submission
  JOURNAL   Exported Friday, Oct 24, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7142
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          join(6310..7142,1..6309)
                     /organism="Cloning vector pWW3536"
                     /mol_type="other DNA"
                     /db_xref="taxon:1977344"
     promoter        1..164
                     /label=PBfP2E5
     terminator      2..44
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     RBS             169..250
                     /label=RBS1P-LP
     CDS             251..865
                     /codon_start=1
                     /label=TetR
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCES"
     terminator      996..1082
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     misc_feature    1083..1122
                     /label=EM_gBlock2
     promoter        1167..1282
                     /label=TetR-56 promoter
     protein_bind    1256..1274
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             1283..1364
                     /label=RBS1P-LP
     CDS             1365..2078
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             2085..2156
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     CDS             2160..2177
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2200..2227
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(2459..3742)
                     /codon_start=1
                     /transl_table=11
                     /product="NBU integrase"
                     /label=NBU integrase
                     /protein_id="ARI71364.1"
                     /translation="MNIKRNIIFALESRKKNGVPIVENVPIRMRVIFASQRIEFTTGYR
                     IDVAKWDADKQRVKSGCTNKLKQSAAEINTDLLKYYAEIQNIFKEFEVQEVMPTTQQLK
                     EAFNMRMKDTSEEQPEEAPVSFWEVFDEFVKECGNQNNWTASTYEKFAAVRNHLKEFKE
                     DATFNYFNEFGLNEYVNFLRDTKDMRNSTIGKQMGFLKWFLRWSFKKGHHQNIAYDTFK
                     PKLKTTSKKVIFLTWDELNKLKDYQIPKDKQYLERVRDVFLFCCFTSLRYSDVRNLKRS
                     DVKSDHIEITTVKTADSLTIELNKYSKAILDKYKDIHFENYMALPVISNQKMNDYLKEL
                     GELAEINEPVRETYYKGNERIDEVTPKYALLSTHAGRRTFICNALALGIPAQVVMKWTG
                     HSDYKAMKPYIDIADDIKANAMNKFNQL"
     CDS             complement(4182..5042)
                     /codon_start=1
                     /transl_table=11
                     /product="AmpR"
                     /label=AmpR
                     /protein_id="ARI71365.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     CDS             5530..6264
                     /codon_start=1
                     /transl_table=11
                     /product="ErmG"
                     /label=ErmG
                     /protein_id="ARI71367.1"
                     /translation="MNKVNIKDSQNFITSKYHIEKIMNCISLDEKDNIFEIGAGKGHFT
                     AGLVKRCNFVTAIEIDSKLCEVTRNKLLNYPNYQIVNDDILKFTFPSHNPYKIFGSIPY
                     NISTNIIRKIVFESSATISYLIVEYGFAKMLLDTNRSLALLLMAEVDISILAKIPRYYF
                     HPKPKVDSTLIVLKRKPAKMAFKERKKYETFVMKWVNKEYEKLFTKNQFNKALKHARIY
                     DINNISFEQFVSLFNSYKIFNG"
     rep_origin      6330..7103
     rep_origin      6334..6722
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     oriT            6893..7002
                     /note="incP origin of transfer"
ORIGIN
        1 gataaaacga aaggctcagt cgaaagactg ggcctttcgt tttacaattg ggctaccttt
       61 tttttgtttt gtttgcaatg gttaatctat tgttaaaatt taaagtttca cttgaacttt
      121 caaataatgt tcttatattt ccagtgtcga aagaaacaaa gtaggactga tcacacaaca
      181 tttaaaaaat aacattatga aagcactcga aaagagattc aaaggtaata ttaacaataa
      241 tttattttca atgtccagat tagataaaag taaagtgatt aacagcgcat tagagctgct
      301 taatgaggtc ggaatcgaag gtttaacaac ccgtaaactc gcccagaagc taggtgtaga
      361 gcagcctaca ttgtattggc atgtaaaaaa taagcgggct ttgctcgacg ccttagccat
      421 tgagatgtta gataggcacc atactcactt ttgcccttta gaaggggaaa gctggcaaga
      481 ttttttacgt aataacgcta aaagttttag atgtgcttta ctaagtcatc gcgatggagc
      541 aaaagtacat ttaggtacac ggcctacaga aaaacagtat gaaactctcg aaaatcaatt
      601 agccttttta tgccaacaag gtttttcact agagaatgca ttatatgcac tcagcgctgt
      661 ggggcatttt actttaggtt gcgtattgga agatcaagag catcaagtcg ctaaagaaga
      721 aagggaaaca cctactactg atagtatgcc gccattatta cgacaagcta tcgaattatt
      781 tgatcaccaa ggtgcagagc cagccttctt attcggcctt gaattgatca tatgcggatt
      841 agaaaaacaa cttaaatgtg aaagtgagac gtaagtggag tgaccttaaa tcaaggacga
      901 tattaatcgg aaggagtatt caacgtgatg aagtcgcagg gttgacgtgg gacgtctctc
      961 ggtataacgc cggaaccgtc ccccgctaga ggcatcaaat aaaacgaaag gctcagtcga
     1021 aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa
     1081 atgcggacaa taaaggatcg tcagaaggta acctgttata caacgggcgg acaataaagg
     1141 atcgtcagaa ggtaacctgt tatacaacgg ttttgttttg tttgcaatgg ttaatctatt
     1201 gttaaaattt aaagtttcac ttgaactttc aaataatgtt cttatatttg cagtgtccct
     1261 atcagtgata gagagtagga ctgatcacac aacatttaaa aaataacatt atgaaagcac
     1321 tcgaaaagag attcaaaggt aatattaaca ataatttatt ttcaatgcgt aaaggcgaag
     1381 agctgttcac tggtgtcgtc cctattctgg tggaactgga tggtgatgtc aacggtcata
     1441 agttttccgt gcgtggcgag ggtgaaggtg acgcaactaa tggtaaactg acgctgaagt
     1501 tcatctgtac tactggtaaa ctgccggttc cttggccgac tctggtaacg acgctgactt
     1561 atggtgttca gtgctttgct cgttatccgg accatatgaa gcagcatgac ttcttcaagt
     1621 ccgccatgcc ggaaggctat gtgcaggaac gcacgatttc ctttaaggat gacggcacgt
     1681 acaaaacgcg tgcggaagtg aaatttgaag gcgataccct ggtaaaccgc attgagctga
     1741 aaggcattga ctttaaagaa gacggcaata tcctgggcca taagctggaa tacaatttta
     1801 acagccacaa tgtttacatc accgccgata aacaaaaaaa tggcattaaa gcgaatttta
     1861 aaattcgcca caacgtggag gatggcagcg tgcagctggc tgatcactac cagcaaaaca
     1921 ctccaatcgg tgatggtcct gttctgctgc cagacaatca ctatctgagc acgcaaagcg
     1981 ttctgtctaa agatccgaac gagaaacgcg atcatatggt tctgctggag ttcgtaaccg
     2041 cagcgggcat cacgcatggt atggatgaac tgtacaaagg atctgattac aaggacgatg
     2101 atgataagga ctacaaagat gatgatgaca aagattataa ggacgatgat gataaaggtc
     2161 atcatcatca tcatcactaa atcctagctg attagctcga gaaggccatc ctgacggatg
     2221 gccttttttt tgtatcgaga cgactagtgt aattgcctat cttccagtga tggaacagca
     2281 tttgtgcatt ggctgcaaca atcagcctta cttgtgcctg ttctatttcc gaaccgaccg
     2341 cttgtatgaa tccatcaaaa ttcgttttct ctatgttgga ttccttgttg ctcatattgt
     2401 gatgataatt tctacaaata tagtcattgg taactatcta tgaaactgtt tgatactttt
     2461 atagttgatt aaacttgttc atggcatttg ccttaatatc atccgctatg tcaatgtagg
     2521 gtttcatagc tttgtagtcg ctgtgtcccg tccatttcat gaccacctgt gccgggattc
     2581 cgagagccag cgcattgcag atgaatgtcc ttcttcctgc atgggtactg agcaaagcgt
     2641 atttgggtgt gacttcatca atacgttcat ttcccttgta gtaggtttcc cgtacaggct
     2701 cgttgatttc tgccagttcg cccagctctt tcaggtaatc gttcatcttc tggttgctga
     2761 tgacgggcag agccatgtaa ttctcgaaat ggatgtcctt gtatttgtcc agtatggctt
     2821 tgctgtattt gttcagttca atcgtcaggc tgtcggcagt cttgactgtg gttatttcga
     2881 tgtggtcgga cttcacatcg cttcttttca gattgcgaac atccgaatac cgcaaactcg
     2941 taaagcagca gaacaggaaa acatcacgca cacgttccag gtattgctta tccttgggta
     3001 tctggtagtc tttcagcttg ttcagttcat cccaagtcag gaagattact tttttcgagg
     3061 tggttttcag tttcggtttg aacgtatcgt atgcaatgtt ctgatgatgt cctttcttga
     3121 agctccagcg caggaaccat ttgaggaatc ccatttgctt gccgatggtg ctgtttctca
     3181 tatccttggt gtcacgcagg aagttgacgt attcgttcaa tccaaactcg ttgaaatagt
     3241 tgaacgttgc atcctccttg aactctttga ggtggttcct cactgctgca aatttttcat
     3301 aggtggatgc cgtccagtta ttctggttac cgcactcttt tacaaactca tcgaacacct
     3361 cccaaaagct gacaggggct tcttccggct gttcttcgct ggtgtctttc attctcatgt
     3421 tgaaagcttc cttcaactgt tgggtcgttg gcatgacctc ctgcacctca aattccttga
     3481 aaatattctg gatttcggca tagtatttca gcaagtccgt attgatttcg gctgcacttt
     3541 gctttagctt gttggtacat ccgctcttta cccgctgctt atctgcatcc catttggcta
     3601 cgtcaatccg gtagcccgtt gtaaactcga tgcgttggct ggcaaagatg acacgcatac
     3661 ggatgggtac gttctctacg attggcacac cgttcttttt ccggctctcc aatgcaaaaa
     3721 tgatgttgcg cttgatattc ataattgggt gcgtttgaaa ttctacaccc aaatatacac
     3781 ccaattattg agatagcaaa agacatttag aaacatttac ttttactcta tattgtaatt
     3841 tacacttgat tatcagtcgt ttgcagtctt atgatattct gtgaaagtat aagttcgaga
     3901 gcctgtctct ccgcaaaaaa cgctgaaaat cagcagattg caaaacaaac accctgtttt
     3961 acacccaaga atgtaaagtc ggctgttttt gttttattta agataataca accactacat
     4021 aataaaagag tagcgatatt aaaagaatcc gatgagaaaa gactaatatt tatctatcca
     4081 ttcagtttga tttttcagga ctttacatcg tcctgaaagt atttgttggt accgaggacg
     4141 cgacagctgc agacgcgtta tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     4201 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     4261 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     4321 cgggacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     4381 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     4441 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     4501 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     4561 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     4621 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     4681 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     4741 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     4801 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     4861 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     4921 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     4981 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     5041 atactcttcc tttttcaatc atggccgcgg gattaaaagt cggggattgg tgaacaaaaa
     5101 ggtgtttctc tctttaagag aaatatcgtt ttgctaaaca gttgatattg aggtatcatt
     5161 ttatcgtaaa agacattttt gctcaacaat tgcttgacgg aaatcaacaa attttagcat
     5221 tttgtaaaaa agtcgctata taatttggtg aattggagtt attttcatat ttttgcatcc
     5281 cgaagagttt ctcttaaaga gagaaacatc ttttgcatac cttttccgac cgaattttta
     5341 tgtcgtaaag aggggctttg cagggggtgg actcagaaag atgagaatag atgactattg
     5401 tagttgaaac acatagaaag ttgctgatat acagaccgat acgcatatcg ggatgaacca
     5461 tgagtacgtt cttttctcaa aaaacataaa tattcgaaaa gagatgcaat aaattaagga
     5521 gaggttataa tgaacaaagt aaatataaaa gatagtcaaa attttattac ttcaaaatat
     5581 cacatagaaa aaataatgaa ttgcataagt ttagatgaaa aagataacat ctttgaaata
     5641 ggtgcaggga aaggtcattt tactgctgga ttggtaaaga gatgtaattt tgtaacggcg
     5701 atagaaattg attctaaatt atgtgaggta actcgtaata agctcttaaa ttatcctaac
     5761 tatcaaatag taaatgatga tatactgaaa tttacatttc ctagccacaa tccatataaa
     5821 atatttggca gcatacctta caacataagc acaaatataa ttcgaaaaat tgtttttgaa
     5881 agttcagcca caataagtta tttaatagtg gaatatggtt ttgctaaaat gttattagat
     5941 acaaacagat cactagcatt gctgttaatg gcagaggtag atatttctat attagcaaaa
     6001 attcctaggt attatttcca tccaaaacct aaagtggata gcacattaat tgtattaaaa
     6061 agaaagccag caaaaatggc atttaaagag agaaaaaaat atgaaacttt tgtaatgaaa
     6121 tgggttaaca aagagtacga aaaactgttt acaaaaaatc aatttaataa agctttaaaa
     6181 catgcgagaa tatatgatat aaacaatatt agtttcgaac aatttgtatc gctatttaat
     6241 agttataaaa tatttaacgg ctaaaaacaa taggccacat gcaactgtaa atgtttacgc
     6301 gggtaccgac accgcggtgg aggggaattc ccatgtcagc cgttaagtgt tcctgtgtca
     6361 ctcaaaattg ctttgagagg ctctaagggc ttctcagtgc gttacatccc tggcttgttg
     6421 tccacaaccg ttaaacctta aaagctttaa aagccttata tattcttttt tttcttataa
     6481 aacttaaaac cttagaggct atttaagttg ctgatttata ttaattttat tgttcaaaca
     6541 tgagagctta gtacgtgaaa catgagagct tagtacgtta gccatgagag cttagtacgt
     6601 tagccatgag ggtttagttc gttaaacatg agagcttagt acgttaaaca tgagagctta
     6661 gtacgtgaaa catgagagct tagtacgtac tatcaacagg ttgaactgct gatcttcaga
     6721 tcctctacgc cggacgcatc gtggccggat caattccgtt ttccgctgca taaccctgct
     6781 tcggggtcat tatagcgatt ttttcggtat atccatcctt tttcgcacga tatacaggat
     6841 tttgccaaag ggttcgtgta gactttcctt ggtgtatcca acggcgtcag ccgggcagga
     6901 taggtgaagt aggcccaccc gcgagcgggt gttccttctt cactgtccct tattcgcacc
     6961 tggcggtgct caacgggaat cctgctctgc gaggctggcc ggctaccgcc ggcgtaacag
     7021 atgagggcaa gcggatggct gatgaaacca agccaaccag gaagggcagc ccacctatca
     7081 cggaattgat ccccctcgaa ttggaattcg ttcctcgatt cagatctaaa ggatccaatc
     7141 ta
//
