LOCUS       Exported                7416 bp DNA     circular SYN 15-JUL-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pFLP2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7416)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7416
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      2..590
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     oriT            660..768
                     /note="incP origin of transfer"
     protein_bind    1152..1173
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        1188..1218
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1226..1242
                     /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     1250..1266
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(1352..2065)
                     /codon_start=1
                     /gene="cIts"
                     /product="temperature-sensitive variant of the phage lambda
                     repressor"
                     /label=lambda repressor (ts)
                     /note="thermosensitivity is conferred by the A67T mutation"
                     /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQ
                     SGVGALFNGINALNAYNAALLTKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMFSPKLRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFG"
     promoter        complement(2072..2101)
                     /label=pRM promoter
     promoter        2106..2155
                     /label=pR promoter
     promoter        2108..2137
                     /label=lambda PR promoter
     RBS             2154..2162
                     /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)"
     CDS             2166..3437
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=FLP
                     /note="FLP is a site-specific recombinase from 
                     Saccharomyces cerevisiae. Recombination occurs at FRT 
                     sequences."
                     /translation="MPQFDILCKTPPKVLVRQFVERFERPSGEKIALCAAELTYLCWMI
                     THNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKKLIPAWEFTI
                     IPYYGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESIWEITEKILN
                     SFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGVIIQCLVTET
                     KTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEYQLLKDNLVR
                     SYNKALKKNAPYSIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWSDKRASAVAR
                     TTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQLKGSAEGSIR
                     YPAWNGIISQEVLDYLSSYINRRI"
     primer_bind     complement(4581..4597)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(4758..5591)
                     /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"
     rep_origin      5605..5956
                     /label=pRO1600 oriV
                     /note="broad-host-range origin of replication from 
                     Pseudomonas aeruginosa plasmid pRO1600; requires the 
                     pRO1600 Rep protein for replication (West et al., 1994)"
     promoter        6282..6386
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6387..7247
                     /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"
ORIGIN
        1 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
       61 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
      121 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
      181 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
      241 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
      301 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
      361 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
      421 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
      481 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
      541 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
      601 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacaag ctagcttatc
      661 ggccagcctc gcagagcagg attcccgttg agcaccgcca ggtgcgaata agggacagtg
      721 aagaaggaac acccgctcgc gggtgggcct acttcaccta tcctgcccgg ctgacgccgt
      781 tggatacacc aaggaaagtc tacacgaacc ctttggcaaa atcctgtata tcgtgcgaaa
      841 aaggatggat ataccgaaaa aatcgctata atgaccccga agcagggtta tgcagcggaa
      901 agtatacctt aaggaatccc catgttcttt cctgcgttat cccctgattc tgtggataac
      961 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     1021 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
     1081 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     1141 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
     1201 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     1261 tatgacatga ttacgaattc gagctcggta ccccgatgaa gattcttgct caattgttat
     1321 cagctatgcg ccgaccagaa caccttgccg atcagccaaa cgtctcttca ggccactgac
     1381 tagcgataac tttccccaca acggaacaac tctcattgca tgggatcatt gggtactgtg
     1441 ggtttagtgg ttgtaaaaac acctgaccgc tatccctgat cagtttcttg aaggtaaact
     1501 catcaccccc aagtctggct atgcagaaat cacctggctc aacagcctgc tcagggtcaa
     1561 cgagaattaa cattccgtca ggaaagcttg gcttggagcc tgttggtgcg gtcatggaat
     1621 taccttcaac ctcaagccag aatgcagaat cactggcttt tttggttgtg cttacccatc
     1681 tctccgcatc acctttggta aaggttctaa gcttaggtga gaacatccct gcctgaacat
     1741 gagaaaaaac agggtactca tactcacttc taagtgacgg ctgcatacta accgcttcat
     1801 acatctcgta gatttctctg gcgattgaag ggctaaattc ttcaacgcta actttgagaa
     1861 tttttgtaag caatgcggcg ttataagcat ttaatgcatt gatgccatta aataaagcac
     1921 caacgcctga ctgccccatc cccatcttgt ctgcgacaga ttcctgggat aagccaagtt
     1981 catttttctt tttttcataa attgctttaa ggcgacgtgc gtcctcaagc tgctcttgtg
     2041 ttaatggttt cttttttgtg ctcatacgtt aaatctatca ccgcaaggga taaatatcta
     2101 acaccgtgcg tgttgactat tttacctctg gcggtgataa tggttgcatg tactaaggag
     2161 gttgtatgcc acaatttgat atattatgta aaacaccacc taaggtgctt gttcgtcagt
     2221 ttgtggaaag gtttgaaaga ccttcaggtg agaaaatagc attatgtgct gctgaactaa
     2281 cctatttatg ttggatgatt acacataacg gaacagcaat caagagagcc acattcatga
     2341 gctataatac tatcataagc aattcgctga gtttcgatat tgtcaataaa tcactccagt
     2401 ttaaatacaa gacgcaaaaa gcaacaattc tggaagcctc attaaagaaa ttgattcctg
     2461 cttgggaatt tacaattatt ccttactatg gacaaaaaca tcaatctgat atcactgata
     2521 ttgtaagtag tttgcaatta cagttcgaat catcggaaga agcagataag ggaaatagcc
     2581 acagtaaaaa aatgcttaaa gcacttctaa gtgagggtga aagcatctgg gagatcactg
     2641 agaaaatact aaattcgttt gagtatactt cgagatttac aaaaacaaaa actttatacc
     2701 aattcctctt cctagctact ttcatcaatt gtggaagatt cagcgatatt aagaacgttg
     2761 atccgaaatc atttaaatta gtccaaaata agtatctggg agtaataatc cagtgtttag
     2821 tgacagagac aaagacaagc gttagtaggc acatatactt ctttagcgca aggggtagga
     2881 tcgatccact tgtatatttg gatgaatttt tgaggaattc tgaaccagtc ctaaaacgag
     2941 taaataggac cggcaattct tcaagcaata aacaggaata ccaattatta aaagataact
     3001 tagtcagatc gtacaataaa gctttgaaga aaaatgcgcc ttattcaatc tttgctataa
     3061 aaaatggccc aaaatctcac attggaagac atttgatgac ctcatttctt tcaatgaagg
     3121 gcctaacgga gttgactaat gttgtgggaa attggagcga taagcgtgct tctgccgtgg
     3181 ccaggacaac gtatactcat cagataacag caatacctga tcactacttc gcactagttt
     3241 ctcggtacta tgcatatgat ccaatatcaa aggaaatgat agcattgaag gatgagacta
     3301 atccaattga ggagtggcag catatagaac agctaaaggg tagtgctgaa ggaagcatac
     3361 gataccccgc atggaatggg ataatatcac aggaggtact agactacctt tcatcctaca
     3421 taaatagacg catataagta cgcatttaag cataaacacg cactatgccg ttcttctcat
     3481 gtatatatat atacaggcaa cacgcagata taggtgcgac gtgaacagtg agctgtatgt
     3541 gcgcagctcg cgttgcattt tcggaagcgc tcgttttcgg aaacgctttg aagttcctat
     3601 tccgaagttc ctattctcta gggggatcct ctagaggatc gatccttttt aacccatcac
     3661 atatacctgc cgttcactat tatttagtga aatgagatat tatgatattt tctgaattgt
     3721 gattaaaaag gcaactttat gcccatgcaa cagaaactat aaaaaataca gagaatgaaa
     3781 agaaacagat agatttttta gttctttagg cccgtagtct gcaaatcctt ttatgatttt
     3841 ctatcaaaca aaagaggaaa atagaccagt tgcaatccaa acgagagtct aatagaatga
     3901 ggtcgaaaag taaatcgcgc gggtttgtta ctgataaagc aggcaaatgg ctaaccgtat
     3961 tcctaacctt ttggtaatga ctccaactta ttgatagtgt tttatgttca gataatgccc
     4021 gatgactttg tcatgcagct ccaccgattt tgagaacgac agcgacttcc gtcccagccg
     4081 tgccaggtgc tgcctcagat tcaggttatg ccgctcaatt cgctgcgtat atcgcttgct
     4141 gattacgtgc agctttccct tcaggcggga ttcatacagc ggccagccat ccgtcatcca
     4201 tatcaccacg tcaaagggtg acagcaggct cataagacgc cccagcgtcg ccatagtgcg
     4261 ttcaccgaat acgtgcgcaa caaccgtctt ccggagactg tcatacgcgt aaaacagcca
     4321 gcgctggcgc gatttagccc cgacatagcc ccactgttcg tccatttccg cgcagacgat
     4381 gacgtcactg cccggctgta tgcgcgaggt taccgactgc ggcctgagtt ttttaagtga
     4441 cgtaaaatcg tgttgaggcc aacgcccata atgcgggctg ttgcccggca tccaacgcca
     4501 ttcatggcca tatcaatgat tttctggtgc gtaccgggtt gagaagcggt gtaagtgaac
     4561 tgcaggcatg caagcttggc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     4621 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     4681 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaaagg caggccgggc
     4741 cgtggtggcc acggcctcta ggccagatcc agcggcatct gggttagtcg agcgcgggcc
     4801 gcttcccatg tctcaccagg gcgagcctgt ttcgcgatct cagcatctga aatcttcccg
     4861 gccttgcgct tcgctggggc cttacccacc gccttggcgg gcttcttcgg tccaaaactg
     4921 aacaacagat gtgtgacctt gcgcccggtc tttcgctgcg cccactccac ctgtagcggg
     4981 ctgtgctcgt tgatctgcgt cacggctgga tcaagcactc gcaacttgaa gtccttgatc
     5041 gagggatacc ggccttccag ttgaaaccac tttcgcagct ggtcaatttc tatttcgcgc
     5101 tggccgatgc tgtcccattg catgagcagc tcgtaaagcc tgatcgcgtg ggtgctgtcc
     5161 atcttggcca cgtcagccaa ggcgtatttg gtgaactgtt tggtgagttc cgtcaggtac
     5221 ggcagcatgt ctttggtgaa cctgagttct acacggccct caccctcccg gtagatgatt
     5281 gtttgcaccc agccggtaat catcacactc ggtcttttcc ccttgccatt gggctcttgg
     5341 gttaaccgga cttcccgccg tttcaggcgc agggccgctt ctttgagctg gttgtaggaa
     5401 gattcgatag ggacacccgc catcgtcgct atgtcctccg ccgtcactga atacatcact
     5461 tcatcggtga caggctcgct cctcttcacc tggctaatac aggccagaac gatccgctgt
     5521 tcctgaacac tgaggcgata cgcggcctcg accagggcat tgcttttgta aaccattggg
     5581 ggtgaggcca cgttcgacat tccttgtgta taaggggaca ctgtatctgc gtcccacaat
     5641 acaacaaatc cgtcccttta caacaacaaa tccgtccctt cttaacaaca aatccgtccc
     5701 ttaatggcaa caaatccgtc cctttttaaa ctctacaggc cacggattac gtggcctgta
     5761 gacgtcctaa aaggtttaaa agggaaaagg aagaaaaggg tggaaacgca aaaaacgcac
     5821 cactacgtgg ccccgttggg gccgcatttg tgcccctgaa ggggcggggg aggcgtctgg
     5881 gcaatccccg ttttaccagt cccctatcgc cgcctgagag ggcgcaggaa gcgagtaatc
     5941 agggtatcga ggcggattca cccttggcgt ccaaccagcg gcaccagcgg cgcctgagag
     6001 gatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
     6061 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     6121 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     6181 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     6241 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     6301 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     6361 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     6421 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     6481 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     6541 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     6601 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     6661 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     6721 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     6781 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     6841 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     6901 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     6961 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     7021 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     7081 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     7141 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     7201 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     7261 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     7321 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     7381 gagcgtcaga ccccgtagaa aagatcaaag gatctt
//
