LOCUS       sequence-494263-        9282 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombineering Plasmid. ITPG-induced expression of Recombitron 
            operon (ccdB dropout cassette in Eco1 Retron); PSP-Tet promoter for 
            Gene III induction upon M13 phage infection and aTc addition..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9282)
  TITLE     Phage-Assisted Evolution of Allosteric Protein Switches
REFERENCE   2  (bases 1 to 9282)
  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"
COMMENT     Sequence Label: RcP9
FEATURES             Location/Qualifiers
     source          1..9282
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(10..748)
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     CDS             complement(823..1905)
                     /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     1867..1886
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1906..1983)
                     /gene="lacI"
                     /label=lacI promoter
     CDS             complement(2088..3362)
                     /codon_start=1
                     /gene="III"
                     /product="pIII"
                     /label=M13 gene III
                     /note="minor coat protein"
                     /translation="MKKLLFAIPLVVPFYSHSAETVESCLAKPHTENSFTNVWKDDKTL
                     DRYANYEGCLWNATGVVVCTGDETQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGG
                     TKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRNRQ
                     GALTVYTGTVTQGTDPVKTYYQYTPVSSKAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQ
                     GQSSDLPQPPVNAGGGSGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGDFDYEKM
                     ANANKGAMTENADENALQSDAKGKLDSVATDYGAAIDGFIGDVSGLANGNGATGDFAGS
                     NSQMAQVGDGDNSPLMNNFRQYLPSLPQSVECRPFVFGAGKPYEFSIDCDKINLFRGVF
                     AFLLYVATFMYVFSTFANILRNKES"
     protein_bind    3432..3450
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      complement(3614..3641)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3733..3819)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3972..3989
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     protein_bind    4060..4076
                     /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             8040..8345
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     8224..8243
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     terminator      8433..8464
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     CDS             complement(8480..9139)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAKCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     9054..9073
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
ORIGIN
        1 ctgcggcggg cgctgcggac acatacaaag ttacccacag attccgtgga taagcagggg
       61 actaacatgt gaggcaaaac agcagggccg cgccggtggc gtttttccat aggctccgcc
      121 ctcctgccag agttcacata aacagacgct tttccggtgc atctgtggga gccgtgaggc
      181 tcaaccatga atctgacagt acgggcgaaa cccgacagga cttaaagatc cccaccgttt
      241 ccggcgggtc gctccctctt gcgctctcct gttccgaccc tgccgtttac cggatacctg
      301 ttccgccttt ctcccttacg ggaagtgtgg cgctttctca tagctcacac actggtatct
      361 cggctcggtg taggtcgttc gctccaagct gggctgtaag caagaactcc ccgttcagcc
      421 cgactgctgc gccttatccg gtaactgttc acttgagtcc aacccggaaa agcacggtaa
      481 aacgccactg gcagcagcca ttggtaactg ggagttcgca gaggatttgt ttagctaaac
      541 acgcggttgc tcttgaagtg tgcgccaaag tccggctaca ctggaaggac agatttggtt
      601 gctgtgctct gcgaaagcca gttaccacgg ttaagcagtt ccccaactga cttaaccttc
      661 gatcaaacca cctccccagg tggttttttc gtttacaggg caaaagatta cgcgcagaaa
      721 aaaaggatct caagaagatc ctttgatctt ttctactgaa ccgctctaga tttcagtgca
      781 atttatctct tcaaatgtag cacctgaagt cagcccagga ggtcactgcc cgctttccag
      841 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
      901 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gacacgggca acagctgatt
      961 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag
     1021 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt
     1081 atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc
     1141 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatacc
     1201 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg
     1261 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag
     1321 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc
     1381 gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat
     1441 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac
     1501 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc
     1561 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac
     1621 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg
     1681 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag
     1741 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc
     1801 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga
     1861 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa
     1921 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt
     1981 gtccgggatc tcgacgctct cccttaagag gacatccggt caaataaaac gaaaggctca
     2041 gtcgaaagac tgggcctttc gttttgctga ggagacttag ggacccttta agactcctta
     2101 ttacgcagta tgttagcaaa cgtagaaaat acatacataa aggtggcaac atataaaaga
     2161 aacgcaaaga caccgcggaa caggttgatc ttatcgcagt cgatactgaa ctcgtaaggt
     2221 ttaccagcgc caaagacaaa agggcgacat tcaaccgatt gagggaggga aggtaaatat
     2281 tgacggaaat tattcattaa aggtgaatta tcaccgtcac cgacttgagc catttgggaa
     2341 ttagagccag caaaatcacc agtagcacca ttaccattag caaggccgga aacgtcacca
     2401 atgaaaccat cgatagcagc accgtaatca gtagcgacag aatcaagttt gcctttagcg
     2461 tcagactgta gcgcgttttc atcggcattt tcggtcatag cccccttatt agcgtttgcc
     2521 atcttttcat aatcaaaatc accggaacca gagccaccac cggaaccgcc tccctcagag
     2581 ccgccaccct cagaaccgcc accctcagag ccaccaccct cagagccgcc accagaacca
     2641 ccaccagagc cgccgccagc attgacagga ggttgaggca ggtcagacga ttggccttga
     2701 tattcacaaa cgaatggatc ctcattaaag ccagaatgga aagcgcagtc tctgaattta
     2761 ccgttccagt aagcgtcata catggctttt gatgatacag gagtgtactg gtaataagtt
     2821 ttaacggggt cagtgccttg agtaacagtg cccgtataaa cagttaatgc cccctgccta
     2881 tttcggaacc tattattctg aaacatgaaa gtattaagag gctgagactc ctcaagagaa
     2941 ggattaggat tagcggggtt ttgctcagta ccaggcggat aagtgccgtc gagagggttg
     3001 atataagtat agcccggaat aggtgtatca ccgtactcag gaggtttagt accgccaccc
     3061 tcagaaccgc caccctcaga accgccaccc tcagagccac caccctcatt ttcagggata
     3121 gcaagcccaa taggaaccca tgtaccgtaa cactgagttt cgtcaccagt acaaactaca
     3181 acgcctgtag cattccacag acagccctca tagttagcgt aacgatctaa agttttgtcg
     3241 tctttccaga cgttagtaaa tgaattttct gtatggggtt ttgctaaaca actttcaaca
     3301 gtttcagcgg agtgagaata gaaaggaaca actaaaggaa ttgcgaataa taattttttc
     3361 attttttttt tcctttactg cacctgcagg taatgttgtc ctcttgattt ctgcgttcag
     3421 gattgtcctg ctctctatca ctgataggga tgaactgtta atacaatttg cgtgccaatt
     3481 ttttatcttt ttgatttata aagatctgat tgaagaatca acagcaacat gccaggatga
     3541 gttagcgaat tacactaaca agtggcgaat ttcatcacgg agccaatgtc ctcagcgagt
     3601 ttgtagaaac gcaaaaaggc catccgtcag gatggccttc tgcttaattt gatgcctggc
     3661 agtttatggc gggcgtcctg cccgccaccc tccgggccgt tgcttcgcaa cgttcaaatc
     3721 cgctcccggc ggatttgtcc tactcaggag agcgttcacc gacaaacaac agataaaacg
     3781 aaaggcccag tctttcgact gagcctttcg ttttatttga tgcctggcag ttccctactc
     3841 tcgcatgggg agaacccaca ctaccatcgg cgctacggcg tttcacttct gagttcggca
     3901 tggggtcagg tgggaccacc gcgctactgc cgccaggcaa attctgtttt atcagaccgc
     3961 ttctgcgttc tgatttaatc tgtatcaggc tgaaaatctt ctctcatccg ccaaaacagc
     4021 cagggcccta ctgataaatg tgagcggata acattgacat tgtgagcgga taacaagata
     4081 ctgagcacat cagcaggacg cactgacctc aacagaaaca ataataatgg agtcatgacc
     4141 atgcctaggc cgcgggccgc gcgaattcag aaggagaata taccatgaac caaatcgtga
     4201 agttcactga cgactctggc ctggcggttc aagttactcc agacgatgtt cgccgttata
     4261 tctgtgagaa cgctactgaa aaagaggtgg gcctctttct gcaactctgt cagactcaac
     4321 gtctgaatcc gtttgtgaaa gacgcttacc tggtgaaata cggcggtgct ccagcttcta
     4381 tgattacttc ctatcaagtt tttaaccgtc gcgcgtgtcg tgatgctaac tatgatggta
     4441 tcaaatctgg tgtggttgtt ctgcgtgacg gtgatgttgt gcataaacgt ggtgctgcgt
     4501 gctacaaaaa ggcgggtgag gagctcatcg gtggttgggc ggaagttcgc tttaaggatg
     4561 gccgcgagac tgcgtatgct gaggtggcgc tcgacgacta ttccaccggc aaatctaatt
     4621 gggcgaaaat gccgggtgtt atgatcgaaa aatgcgcgaa ggctgctgct tggcgcctcg
     4681 cgttcccgga cacttttcag ggcatgtacg ctgcggagga aatggatcaa gcgcaacagc
     4741 cagaacaggt gcgcgctcag gcggagcaac cagtggatct ccagccaatc cgcgaactct
     4801 tcaagccata ttgcgaacac ttcggcatca ctccggctga gggtatgact gctgtttgtg
     4861 gtgcggtggg cgctgaaggc atgcactcta tgaccgagca gcaagctcgc cgtgctcgcg
     4921 cttggatgga ggaagaaatg gctgcgccag ctgtggaagc ggagtatgag gttgttgacg
     4981 agggcgaggt gttttaagga tccaggagaa tacacatgcc aattcaggtc ttaccgccac
     5041 aactggcgaa ccagattgcc gcaggtgagg tggtcgagcg acctgcgtcg gtagtcaaag
     5101 aactagtgaa aaacagcctc gatgcaggtg cgacgcgtat cgatattgat atcgaacgcg
     5161 gtggggcgaa acttatccgc attcgtgata acggctgcgg tatcaaaaaa gatgagctgg
     5221 cgctggcgct ggctcgtcat gccaccagta aaatcgcctc tctggacgat ctcgaagcca
     5281 ttatcagcct gggctttcgc ggtgaggcgc tggcgagtat cagttcggtt tcccgcctga
     5341 cgctcacttc acgcaccgca gaacagcagg aagcctggca ggcctatgcc gaagggcgcg
     5401 atatgaacgt gacggtaaaa ccggcggcgc atcctgtggg gacgacgctg gaggtgctgg
     5461 atctgttcta caacaccccg gcgcggcgca aattcctgcg caccgagaaa accgaattta
     5521 accacattga tgagatcatc cgccgcattg cgctggcgcg tttcgacgtc acgatcaacc
     5581 tgtcgcataa cggtaaaatt gtgcgtcagt accgcgcagt gccggaaggc gggcaaaaag
     5641 aacggcgctt aggcgcgatt tgcggcaccg cttttcttga acaagcgctg gcgattgaat
     5701 ggcaacacgg cgatctcacg ctacgcggct gggtggccga tccaaatcac accacgcccg
     5761 cactggcaga aattcagtat tgctacgtga acggtcgcat gatgcgcgat cgcctgatca
     5821 atcacgcgat ccgccaggcc tgcgaagaca aactgggggc cgatcagcaa ccggcatttg
     5881 tgttgtatct ggagatcgac ccacatcagg tggacgtcaa cgtgcacccc gccaaacacg
     5941 aagtgcgttt ccatcagtcg cgtctggtgc atgattttat ctatcagggc gtgctgagcg
     6001 tgctacaaca gcaactggaa acgccgctac cgctggacga tgaaccccaa cctgcaccgc
     6061 gttccattcc ggaaaaccgc gtggcggcgg ggcgcaatca ctttgcagaa ccggcagctc
     6121 gtgagccggt agctccgcgc tacactcctg cgccagcatc aggcagtcgt ccggctgccc
     6181 cctggccgaa tgcgcagcca ggctaccaga aacagcaagg tgaagtgtat cgccagcttt
     6241 tgcaaacgcc cgcgccgatg caaaaattaa aagcgccgga accgcaggaa cctgcacttg
     6301 cggcgaacag tcagagtttt ggtcgggtac tgactatcgt ccattccgac tgtgcgttgc
     6361 tggagcgcga cggcaacatt tcacttttat ccttgccagt ggcagaacgt tggctgcgtc
     6421 aggcacaatt gacgccgggt gaagcgcccg tttgcgccca gccgctgctg attccgttgc
     6481 ggctaaaagt ttctgccgaa gaaaaatcgg cattagaaaa agcgcagtct gccctggcgg
     6541 aattgggtat tgatttccag tcagatgcac agcatgtgac catcagggca gtgcctttac
     6601 ccttacgcca acaaaattta caaatcttga ttcctgaact gataggctac ctggcgaagc
     6661 agtccgtatt cgaacctggc aatattgcgc agtggattgc acgaaatctg atgagcgaac
     6721 atgcgcagtg gtcaatggca caggccataa ccctgctggc ggacgtggaa cggttatgtc
     6781 cgcaacttgt gaaaacgccg ccgggtggtc tgttacaatc tgttgattta catccggcga
     6841 taaaagccct gaaagatgag tgaaaaggaa aaaaaaatga aatccgctga atatttgaac
     6901 acttttagat tgagaaatct cggcctacct gtcatgaaca atttgcatga catgtctaag
     6961 gcgactcgca tatctgttga aacacttcgg ttgttaatct atacagctga ttttcgctat
     7021 aggatctaca ctgtagaaaa gaaaggccca gagaagagaa tgagaaccat ttaccaacct
     7081 tctcgagaac ttaaagcctt acaaggatgg gttctacgta acattttaga taaactgtcg
     7141 tcatctcctt tttctattgg atttgaaaag caccaatcta ttttgaataa tgctaccccg
     7201 catattgggg caaactttat actgaatatt gatttggagg attttttccc aagtttaact
     7261 gctaacaaag tttttggagt gttccattct cttggttata atcgactaat atcttcagtt
     7321 ttgacaaaaa tatgttgtta taaaaatctg ctaccacaag gtgctccatc atcacctaaa
     7381 ttagctaatc taatatgttc taaacttgat tatcgtattc agggttatgc aggtagtcgg
     7441 ggcttgatat atacgagata tgccgatgat ctcaccttat ctgcacagtc tatgaaaaag
     7501 gttgttaaag cacgtgattt tttattttct ataatcccaa gtgaaggatt ggttattaac
     7561 tcaaaaaaaa cttgtattag tgggcctcgt agtcagagga aagttacagg tttagttatt
     7621 tcacaagaga aagttgggat aggtagagaa aaatataaag aaattagagc aaagatacat
     7681 catatatttt gcggtaagtc ttctgagata gaacacgtta ggggatggtt gtcatttatt
     7741 ttaagtgtgg attcaaaaag ccataggaga ttaataactt atattagcaa attagaaaaa
     7801 aaatatggaa agaacccttt aaataaagcg aagacctaac atttaactat gataagattc
     7861 cgtatgcgca cccttagcga gaggtttatc attaaggtca acctctggat gttgtttcgg
     7921 catcctgcat tgaatctgag ttactgtctg ttttcctgaa ttctgagacg tggcacaccc
     7981 cttgctttgt ctttatcaac gcaaataaca agttgataac aagctagcag gaggaattca
     8041 tgcagtttaa ggtttacacc tataaaagag agagccgtta tcgtctgttt gtggatgtac
     8101 agagtgatat tattgacacg cccgggcgac ggatggtgat ccccctggcc agtgcacgtc
     8161 tgctgtcaga taaagtctcc cgtgaacttt acccggtggt gcatatcggg gatgaaagct
     8221 ggcgcatgat gaccaccgat atggccagtg tgccggtctc cgttatcggg gaagaagtgg
     8281 ctgatctcag ccaccgcgaa aatgacatca aaaacgccat taacctgatg ttctggggaa
     8341 tataacgtct cagtacgaat tcaggaaacc cgttttttct gacgtaaggg tgcgcatacg
     8401 gaatctactt aattaacggc actcctcagc caagtcaaaa gcctccgacc ggaggctttt
     8461 gactacatgc ccatggcgtt tacgccccgc cctgccactc atcgcagtac tgttgtaatt
     8521 cattaagcat tctgccgaca tggaagccat cacaaacggc atgatgaacc tgaatcgcca
     8581 gcggcatcag caccttgtcg ccttgcgtat aatatttgcc catagtgaaa acgggggcga
     8641 agaagttgtc catattggcc acgtttaaat caaaactggt gaaactcacc cagggattgg
     8701 ctgaaacgaa aaacatattc tcaataaacc ctttagggaa ataggccagg ttttcaccgt
     8761 aacacgccac atcttgcgaa tatatgtgta gaaactgccg gaaatcgtcg tggtattcac
     8821 tccagagcga tgaaaacgtt tcagtttgct catggaaaac ggtgtaacaa gggtgaacac
     8881 tatcccatat caccagctca ccgtctttca ttgccatacg gaactccgga tgagcattca
     8941 tcaggcgggc aagaatgtga ataaaggccg gataaaactt gtgcttattt ttctttacgg
     9001 tctttaaaaa ggccgtaata tccagctgaa cggtctggtt ataggtacat ttagcaactg
     9061 actgaaatgc ctcaaaatgt tctttacgat gccattggga tatatcaacg gtggtatatc
     9121 cagtgatttt tttctccatt ttagcttcct tagctcctga aaatctcgat aactcaaaaa
     9181 atacgcccgg tagtgatctt atttcattat ggtgaaagtt ggaacctctt acgtgccaag
     9241 ccaaataggc cgtcactcgg tcgctacgct ccgggcgtga ga
//
