LOCUS       sequence-367523-        6435 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Broad host range regulated expression of the DddA with a C-terminal 
            fusion to a FLAG epitope (DddAI-FLAG).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6435)
  AUTHORS   Gallagher LA, Velazquez E, Peterson SB, Charity JC, Radey MC, 
            Gebhardt MJ, Hsu F, Shull LM, Cutler KJ, Macareno K, de Moraes MH, 
            Penewit KM, Kim J, Andrade PA, LaFramboise T, Salipante SJ, Reniere 
            ML, de Lorenzo V, Wiggins PA, Dove SL, Mougous JD
  TITLE     Genome-wide protein-DNA interaction site mapping in bacteria using a
            double-stranded DNA-specific cytosine deaminase.
  JOURNAL   Nat Microbiol. 2022 Jun;7(6):844-855. doi: 
            10.1038/s41564-022-01133-9. Epub 2022 Jun 1.
  PUBMED    35650286
REFERENCE   2  (bases 1 to 6435)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41564-022-01133-9"; journalName: "Nat Microbiol"; date: 
            "2022-06"; volume: "7"; issue: "6"; pages: "844-855"
COMMENT     Sequence Label: pPSV39-dddAI-FLAG
FEATURES             Location/Qualifiers
     source          1..6435
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(11..16,17..34,35..41)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    49..65
                     /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)."
     CDS             464..487
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             498..530
                     /codon_start=1
                     /product="epitope tag from vesicular stomatitis virus G 
                     protein"
                     /label=VSV-G tag
                     /translation="YTDIEMNRLGK"
     terminator      564..610
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(899..917)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             1144..2226
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(1163..1182)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(2270..2275,2276..2292,2293..2298)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             2487..3020
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     oriT            3147..3256
                     /note="incP origin of transfer"
     rep_origin      3648..4236
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4137..4156
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4390..4407
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4422..4562
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(4664..4855)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5373..6206)
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pRO1600 from Pseudomonas aeruginosa "
                     /label=pRO1600 Rep
                     /translation="MASPPMVYKSNALVEAAYRLSVQEQRIVLACISQVKRSEPVTDEV
                     MYSVTAEDIATMAGVPIESSYNQLKEAALRLKRREVRLTQEPNGKGKRPSVMITGWVQT
                     IIYREGEGRVELRFTKDMLPYLTELTKQFTKYALADVAKMDSTHAIRLYELLMQWDSIG
                     QREIEIDQLRKWFQLEGRYPSIKDFKLRVLDPAVTQINEHSPLQVEWAQRKTGRKVTHL
                     LFSFGPKKPAKAVGKAPAKRKAGKISDAEIAKQARPGETWEAARARLTQMPLDLA"
ORIGIN
        1 cactacgtgc tttacacttt atgcttccgg ctcgtataat gtgtggaatt gtgagcggat
       61 aacaatttca gaattcgagc tcacgggagg aaagatgtac gcagacgatt tcgacggcga
      121 aattgagatc gatgaggttg atagcttggt cgagttcctc agcagaagac ctgcatttga
      181 cgcaaataat tttgttttga cgtttgagga aagtggtttt cctcaattga atatttttgc
      241 aaaaaacgat atcgccgttg tttattatat ggacatcggc gagaatttcg tttcgaaagg
      301 caattctgcg tcaggtggta cggagaagtt ttacgagaat aagcttggcg gggaagtaga
      361 cctgtcgaaa gattgcgttg tgagtaaaga gcaaatgatt gaagcggcaa agcagttttt
      421 tgctacgaag caacggcccg agcaactgac atggagcgag ttggactaca aagacgacga
      481 cgacaaatga tctagaatat acagatattg aaatgaatag attaggaaaa tgagctagag
      541 tcgacctgca ggcatgcaag ctttagcata accccttggg gcctctaaac gggtcttgag
      601 gggttttttg atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcctgatg
      661 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt
      721 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
      781 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
      841 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
      901 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
      961 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     1021 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattaatgg
     1081 agcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg
     1141 aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc
     1201 gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa
     1261 gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa
     1321 cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt
     1381 gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta
     1441 gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc
     1501 agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc
     1561 tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt
     1621 attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac
     1681 cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct
     1741 ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac
     1801 tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc
     1861 actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag
     1921 tccgggctgc gcgttggtgc ggatatctcg gtagtgggat acgacgatac cgaagacagc
     1981 tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc
     2041 agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg
     2101 cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgcctctccc
     2161 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     2221 cagtgagcgc aacgcaatta atgtgagaat attacgcgta attctcgaat tgacataagc
     2281 ctgttcggtt cgtaaactgt aatgcaagta gcgtatgcgc tcacgcaact ggtccagaac
     2341 cttgaccgaa cgcagcggtg gtaacggcgc agtggcggtt ttcatggctt gttatgactg
     2401 tttttttgta cagtctatgc ctcgggcatc caagcagcaa gcgcgttacg ccgtgggtcg
     2461 atgtttgatg ttatggagca gcaacgatgt tacgcagcag caacgatgtt acgcagcagg
     2521 gcagtcgccc taaaacaaag ttaggtggct caagtatggg catcattcgc acatgtaggc
     2581 tcggccctga ccaagtcaaa tccatgcggg ctgctcttga tcttttcggt cgtgagttcg
     2641 gagacgtagc cacctactcc caacatcagc cggactccga ttacctcggg aacttgctcc
     2701 gtagtaagac attcatcgcg cttgctgcct tcgaccaaga agcggttgtt ggcgctctcg
     2761 cggcttacgt tctgcccaag tttgagcagc cgcgtagtga gatctatatc tatgatctcg
     2821 cagtctccgg cgagcaccgg aggcagggca ttgccaccgc gctcatcaat ctcctcaagc
     2881 atgaggccaa cgcgcttggt gcttatgtga tctacgtgca agcagattac ggtgacgatc
     2941 ccgcagtggc tctctataca aagttgggca tacgggaaga agtgatgcac tttgatatcg
     3001 acccaagtac cgccacctaa caattcgttc aagccgagat cggcttcccg gccgacgcgt
     3061 caattccgtg ataggtgggc tgcccttcct ggttggcttg gtttcatcag ccatccgctt
     3121 gccctcatct gttacgccgg cggtagccgg ccagcctcgc agagcaggat tcccgttgag
     3181 caccgccagg tgcgaataag ggacagtgaa gaaggaacac ccgctcgcgg gtgggcctac
     3241 ttcacctatc ctgcccggct gacgccgttg gatacaccaa ggaaagtcta cacgaaccct
     3301 ttggcaaaat cctgtatatc gtgcgaaaaa ggatggatat accgaaaaaa tcgctataat
     3361 gaccccgaag cagggttatg cagcggaaaa cggaattgga cggggagtca ggcaactatg
     3421 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     3481 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     3541 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     3601 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     3661 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     3721 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     3781 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     3841 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     3901 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     3961 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4021 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4081 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4141 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4201 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     4261 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     4321 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     4381 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     4441 cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct
     4501 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     4561 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     4621 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     4681 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
     4741 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     4801 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
     4861 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
     4921 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
     4981 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     5041 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     5101 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga
     5161 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     5221 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     5281 gccgggtcct caacgacagg agcacgatca tgcgcacccg tggaaattaa ttaattgcag
     5341 aaaggcaggc cgggccgtgg tggccacggc ctctaggcca gatccagcgg catctgggtt
     5401 agtcgagcgc gggccgcttc ccatgtctca ccagggcgag cctgtttcgc gatctcagca
     5461 tctgaaatct tcccggcctt gcgcttcgct ggggccttac ccaccgcctt ggcgggcttc
     5521 ttcggtccaa aactgaacaa cagatgtgtg accttgcgcc cggtctttcg ctgcgcccac
     5581 tccacctgta gcgggctgtg ctcgttgatc tgcgtcacgg ctggatcaag cactcgcaac
     5641 ttgaagtcct tgatcgaggg ataccggcct tccagttgaa accactttcg cagctggtca
     5701 atttctattt cgcgctggcc gatgctgtcc cattgcatga gcagctcgta aagcctgatc
     5761 gcgtgggtgc tgtccatctt ggccacgtca gccaaggcgt atttggtgaa ctgtttggtg
     5821 agttccgtca ggtacggcag catgtctttg gtgaacctga gttctacacg gccctcaccc
     5881 tcccggtaga tgattgtttg cacccagccg gtaatcatca cactcggtct tttccccttg
     5941 ccattgggct cttgggttaa ccggacttcc cgccgtttca ggcgcagggc cgcttctttg
     6001 agctggttgt aggaagattc gatagggaca cccgccatcg tcgctatgtc ctccgccgtc
     6061 actgaataca tcacttcatc ggtgacaggc tcgctcctct tcacctggct aatacaggcc
     6121 agaacgatcc gctgttcctg aacactgagg cgatacgcgg cctcgaccag ggcattgctt
     6181 ttgtaaacca ttgggggtga ggccacgttc gacattcctt gtgtataagg ggacactgta
     6241 tctgcgtccc acaatacaac aaatccgtcc ctttacaaca acaaatccgt cccttcttaa
     6301 caacaaatcc gtcccttaat ggcaacaaat ccgtcccttt ttaaactcta caggccacgg
     6361 attacgtggc ctgtagacgt cctaaaaggt ttaaaaggga aaaggaagaa aagggtggaa
     6421 acgcaaaaaa cgcac
//
