LOCUS       sequence-148741-        6591 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Input plasmid: expresses two recombinases downstream of two 
            inducible promoters. Same as Dual-Recombinase-Controller (plasmid 
            #44456) from Endy Lab except with KanR instead of CamR on the 
            backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6591)
  AUTHORS   Roquet N, Soleimany AP, Ferris AC, Aaronson S, Lu TK
  TITLE     Synthetic recombinase-based state machines in living cells.
  JOURNAL   Science. 2016 Jul 22;353(6297):aad8559. doi: 
            10.1126/science.aad8559. Epub 2016 Jul 21.
  PUBMED    27463678
REFERENCE   2  (bases 1 to 6591)
  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"; journalName: "Science."; 
            date: "2016-07-22"; volume: "353(6297):aad8559. doi"; pages: " 
            10.1126/science.aad8559. Epub 2016 Jul 21"
COMMENT     Sequence Label: pNR64
FEATURES             Location/Qualifiers
     source          1..6591
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(156..744)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(236..255)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(832..926)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(957..1751)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1068..1087)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1678..1697
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     misc_feature    1906..1927
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     protein_bind    1928..1946
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1953..1971
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             2016..3515
                     /codon_start=1
                     /transl_table=11
                     /gene="Bxb1"
                     /product="Bxb1 integrase"
                     /label=Bxb1 integrase
                     /note="Bxb1 large serine integrase from Mycobacterium 
                     bacteriophage Bxb1 (Ghosh et al. 2003)."
                     /protein_id="AFF61396.1"
                     /translation="RALVVIRLSRVTDATTSPERQLESCQQLCAQRGWDVVGVAEDLDV
                     SGAVDPFDRKRRPNLARWLAFEEQPFDVIVAYRVDRLTRSIRHLQQLVHWAEDHKKLVV
                     SATEAHFDTTTPFAAVVIALMGTVAQMELEAIKERNRSAAHFNIRAGKYRGSLPPWGYL
                     PTRVDGEWRLVPDPVQRERILEVYHRVVDNHEPLHLVAHDLNRRGVLSPKDYFAQLQGR
                     EPQGREWSATALKRSMISGAMLGYATLNGKTVRDDDGAPLVRAEPILTREQLEALRAEL
                     VKTSRAKPAVSTPSLLLRVLFCAVCGEPAYKFAGGGRKHPRYRCRSMGFPKHCGNGTVA
                     MAEWDAFCEEQVLDLLGDAERLEKVWVAGSDSAVELAEVNAELVDLTSLIGSPAYRAGS
                     PQREALDARIAALAARQEELEGLEARPSGWEWRETGQRFGDWWREQDTAAKNTWLRSMN
                     VRLTFDVRGGLTRTIDFGDLQEYEQHLRLGSVVERLHTGMS"
     terminator      3544..3571
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(3654..4532)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWAYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        4559..4843
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     4766..4785
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             4897..4914
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6382..6414
                     /codon_start=1
                     /product="C-terminal peptide that mediates degradation in 
                     bacteria through the ClpXP and ClpAP proteases (McGinness 
                     et al., 2006)"
                     /label=ssrA tag
                     /note="wild-type LAA variant"
                     /translation="AAKDENYALAA"
     misc_feature    6418..6438
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      6439..6496
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     primer_bind     complement(6520..6537)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cctagctagg gcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
       61 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
      121 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
      181 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
      241 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
      301 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
      361 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
      421 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
      481 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
      541 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
      601 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
      661 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
      721 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
      781 cagtggaacg aaaactcacg ttaagggatt ttggtcatga ctagtgcttg gattctcacc
      841 aataaaaaac gcccggcggc aaccgagcgt tctgaacaaa tccagatgga gttctgaggt
      901 cattactgga tctatcaaca ggagtccaag cgagctctcg aaccccagag tcccgctcag
      961 aagaactcgt caagaaggcg atagaaggcg atgcgctgcg aatcgggagc ggcgataccg
     1021 taaagcacga ggaagcggtc agcccattcg ccgccaagct cttcagcaat atcacgggta
     1081 gccaacgcta tgtcctgata gcggtccgcc acacccagcc ggccacagtc gatgaatcca
     1141 gaaaagcggc cattttccac catgatattc ggcaagcagg catcgccatg ggtcacgacg
     1201 agatcctcgc cgtcgggcat gcgcgccttg agcctggcga acagttcggc tggcgcgagc
     1261 ccctgatgct cttcgtccag atcatcctga tcgacaagac cggcttccat ccgagtacgt
     1321 gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc aggtagccgg atcaagcgta
     1381 tgcagccgcc gcattgcatc agccatgatg gatactttct cggcaggagc aaggtgagat
     1441 gacaggagat cctgccccgg cacttcgccc aatagcagcc agtcccttcc cgcttcagtg
     1501 acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg ccagccacga tagccgcgct
     1561 gcctcgtcct gcagttcatt cagggcaccg gacaggtcgg tcttgacaaa aagaaccggg
     1621 cgcccctgcg ctgacagccg gaacacggcg gcatcagagc agccgattgt ctgttgtgcc
     1681 cagtcatagc cgaatagcct ctccacccaa gcggccggag aacctgcgtg caatccatct
     1741 tgttcaatca tgcgaaacga tcctcatcct gtctcttgat cagatcttga tcccctgcgc
     1801 catcagatcc ttggcggcaa gaaagccatc cagtttactt tgcagggctt cccaacctta
     1861 ccagagggcg ccccagctgg caattccgac gtcaaagggc ccagagaatt cgcggccgct
     1921 tctagagtcc ctatcagtga tagagattga catccctatc agtgatagag atactgagca
     1981 caagcttgcg gctttcacac ataggagcta gcgtgagagc cctggtagtc atccgcctgt
     2041 cccgcgtcac cgatgctacg acttcaccgg agcgtcagct ggagtcttgc cagcagctct
     2101 gcgcccagcg cggctgggac gtcgtcgggg tagcggagga tctggacgtc tccggggcgg
     2161 tcgatccgtt cgaccggaag cgcagaccga acctggcccg gtggctagcg ttcgaggagc
     2221 aaccgtttga cgtgatcgtg gcgtaccggg tagatcggtt gacccgatcg atccggcatc
     2281 ttcagcagct ggtccactgg gccgaggacc acaagaagct ggtcgtctcc gcgaccgaag
     2341 cgcacttcga tacgacgacg ccgtttgcgg cggtcgtcat cgcgcttatg ggaacggtgg
     2401 cgcagatgga attagaagcg atcaaagagc ggaaccgttc ggctgcgcat ttcaatatcc
     2461 gcgccgggaa ataccgagga tccctgccgc cgtggggata cctgcctacg cgcgtggacg
     2521 gggagtggcg gctggtgccg gaccctgtgc agcgagagcg catcctcgag gtgtatcacc
     2581 gcgtcgtcga caaccacgag ccgctgcatc tggtggccca cgacctgaac cggcgtggtg
     2641 tcctgtcgcc gaaggactac ttcgcgcagc tgcaaggccg cgagccgcag ggccgggagt
     2701 ggtcggctac cgcgctgaag cgatcgatga tctccggggc gatgctcggg tacgcgactc
     2761 tgaacggtaa gaccgtccga gacgacgacg gagccccgct ggtgcgggct gagccgatcc
     2821 tgacccgtga gcagctggag gcgctgcgcg ccgagctcgt gaagacctcc cgggcgaagc
     2881 ccgcggtgtc taccccgtcg ctgctgctgc gggtgttgtt ctgcgcggtg tgcggggagc
     2941 ccgcgtacaa gttcgccggg ggaggacgta agcacccgcg ctaccgctgc cgctcgatgg
     3001 ggttcccgaa gcactgcggg aacggcacgg tggcgatggc cgagtgggac gcgttctgcg
     3061 aggagcaggt actggatctg ctcggggacg cggagcgtct ggagaaagtc tgggtagcgg
     3121 gctcggactc cgcggtcgaa ctcgcggagg tgaacgcgga gctggtggac ctgacgtcgc
     3181 tgatcggctc cccggcctac cgggcgggct ctccgcagcg agaagcactg gatgcccgta
     3241 ttgcggcgct ggccgcgcgg caagaggagc tggagggcct ggaggctcgc ccgtctggct
     3301 gggagtggcg cgagaccggg cagcggttcg gggactggtg gcgggagcag gacaccgcgg
     3361 caaagaacac ctggcttcgg tcgatgaacg ttcggctgac gttcgacgtc cgcggcgggc
     3421 tgactcgcac gatcgacttc ggggatcttc aggagtacga gcagcatctc aggctcggca
     3481 gcgtggtcga acggctacac accgggatgt cgtaataata ctagatgcat taataatcac
     3541 actggctcac cttcgggtgg gcctttctgc gtttatatac tagagagaga atataaaaag
     3601 ccagattatt aatccggctt ttttattatt tcctgcaggg cggccgccat atgttatgac
     3661 aacttgacgg ctacatcatt cactttttct tcacaaccgg cacggaactc gctcgggctg
     3721 gccccggtgc attttttaaa tacccgcgag aaatagagtt gatcgtcaaa accaacattg
     3781 cgaccgacgg tggcgatagg catccgggtg gtgctcaaaa gcagcttcgc ctggctgata
     3841 cgttggtcct cgcgccagct taagacgcta atccctaact gctggcggaa aagatgtgac
     3901 agacgcgacg gcgacaagca aacatgctgt gcgacgctgg cgatatcaaa attgctgtct
     3961 gccaggtgat cgctgatgta ctgacaagcc tcgcgtaccc gattatccat cggtggatgg
     4021 agcgactcgt taatcgcttc catgcgccgc agtaacaatt gctcaagcag atttatcgcc
     4081 agcagctccg aatagcgccc ttccccttgc ccggcgttaa tgatttgccc aaacaggtcg
     4141 ctgaaatgcg gctggtgcgc ttcatccggg cgaaagaacc ccgtattggc aaatattgac
     4201 ggccagttaa gccattcatg ccagtaggcg cgcggacgaa agtaagccca ctggtgatac
     4261 cattcgcgag cctccggatg acgaccgtag tgatgaatct ctcctggcgg gaacagcaaa
     4321 atatcacccg gtcggcaaac aaattctcgt ccctgatttt tcaccacccc ctgaccgcga
     4381 atggtgagat tgagaatata acctttcatt cccagcggtc ggtcgataaa aaaatcgaga
     4441 taaccgttgg cctcaatcgg cgttaaaccc gccaccagat gggcattaaa cgagtatccc
     4501 ggcagcaggg gatcattttg cgcttcagcc atacttttca tactcccgcc attcagagaa
     4561 gaaaccaatt gtccatattg catcagacat tgccgtcact gcgtctttta ctggctcttc
     4621 tcgctaacca aaccggtaac cccgcttatt aaaagcattc tgtaacaaag cgggaccaaa
     4681 gccatgacaa aaacgcgtaa caaaagtgtc tataatcacg gcagaaaagt ccacattgat
     4741 tatttgcacg gcgtcactct ttgctatgcc atagcatttt tatccataag attagcggat
     4801 cctacctgac gctttttatc gcaactctct actgtttctc cataccgttt ttttgggcta
     4861 gctactagag tcacacagga aacctactag atgaaacatc atcaccatca ccaccaggcc
     4921 ggcactaaga aagtagcaat ctatacacga gtatccacta ctaaccaagc agaggaaggc
     4981 ttctcaattg atgagcaaat tgaccgttta acaaaatatg ctgaagcaat ggggtggcaa
     5041 gtatctgata cttatactga tgctggtttt tcaggggcca aacttgaacg cccagcaatg
     5101 caaagattaa tcaacgatat cgagaataaa gcttttgata cagttcttgt atataagcta
     5161 gaccgccttt cacgtagtgt aagagatact ctttatcttg ttaaggatgt gttcacaaaa
     5221 aataaaatag actttatctc gcttaatgaa agtattgata cttcttctgc tatgggtagc
     5281 ttgtttctca ctattctttc tgcaattaat gagtttgaaa gagagaatat aaaagaacgc
     5341 atgactatgg gtaaactagg gcgagcgaaa tctggtaagt ctatgatgtg gactaagaca
     5401 gcttttgggt attaccacaa cagaaagaca ggtatattag aaattgttcc tttacaagct
     5461 acaatagttg aacaaatatt cactgattat ttatcaggaa tatcacttac aaaattaaga
     5521 gataaactca atgaatctgg acacatcggt aaagatatac cgtggtctta tcgtacccta
     5581 agacaaacac ttgataatcc agtttactgt ggttatatca aatttaagga cagcctattt
     5641 gaaggtatgc acaaaccaat tatcccttat gagacttatt taaaagttca aaaagagcta
     5701 gaagaaagac aacagcagac ttatgaaaga aataacaacc ctagaccttt ccaagctaaa
     5761 tatatgctgt cagggatggc aaggtgcggt tactgtggag cacctttaaa aattgttctt
     5821 ggccacaaaa gaaaagatgg aagccgcact atgaaatatc actgtgcaaa tagatttcct
     5881 cgaaaaacaa aaggaattac agtatataat gacaataaaa agtgtgattc aggaacttat
     5941 gatttaagta atttagaaaa tactgttatt gacaacctga ttggatttca agaaaataat
     6001 gactccttat tgaaaattat caatggcaac aaccaaccta ttcttgatac ttcgtcattt
     6061 aaaaagcaaa tttcacagat cgataaaaaa atacaaaaga actctgattt gtacctaaat
     6121 gattttatca ctatggatga gttgaaagat cgtactgatt cccttcaggc tgagaaaaag
     6181 ctgcttaaag ctaagattag cgaaaataaa tttaatgact ctactgatgt ttttgagtta
     6241 gttaaaactc agttgggctc aattccgatt aatgaactat catatgataa taaaaagaaa
     6301 atcgtcaaca accttgtatc aaaggttgat gttactgctg ataatgtaga tatcatattt
     6361 aaattccaac tcgctaccgg tgctgctaag gacgaaaact acgctctggc tgcttaatac
     6421 tagtagcggc cgctgcagtc cggcaaaaaa gggcaaggtg tcaccaccct gccctttttc
     6481 tttaaaaccg aaaagattac ttcgcgttat gcaggcttcc tcgctcactg actcgctgcg
     6541 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa t
//
