LOCUS       sequence-119852-        7481 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Clone cassette to replace RFP for integration into C31 landing pad 
            strain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7481)
  AUTHORS   Heil JR, Cheng J, Charles TC
  TITLE     Site-specific bacterial chromosome engineering: PhiC31 integrase 
            mediated cassette exchange (IMCE).
  JOURNAL   J Vis Exp. 2012 Mar 16;(61). pii: 3698. doi: 10.3791/3698.
  PUBMED    22453661
REFERENCE   2  (bases 1 to 7481)
  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: "J Vis 
            Exp."; date: "2012-03-16"; volume: "(61). pii: 3698. doi"; pages: " 
            10.3791/3698"
COMMENT     Sequence Label: pJH110
FEATURES             Location/Qualifiers
     source          1..7481
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(20..25,26..43,44..50)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    58..74
                     /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     63..85
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     82..98
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     82..98
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    117..150
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     CDS             complement(290..823)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPRFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(1012..1017,1018..1034,1035..1040))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             complement(1116..1793)
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     RBS             1800..1811
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        complement(1820..1854)
                     /label=BBa_J23118
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.56"
     protein_bind    1861..1894
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     primer_bind     complement(1929..1946)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1929..1945)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1938..1960)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             2450..3244
                     /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(2504..2523)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3114..3133
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     promoter        3290..3735
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3736..3822,3823..5157)
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVSEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     oriT            5511..5620
                     /note="incP origin of transfer"
     promoter        complement(5855..5945)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     promoter        complement(6139..6229)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      6589..7177
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7078..7097
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7331..7348
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    join(7465..7481,1..5)
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
       61 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aagcttgtgc
      121 cagggcgtgc ccttgggctc cccgggcgcg tgaaataagt ggctgcgcgc ataaagcggg
      181 tgatgggact aacgcctgag ttaagccgga gcgctttgcg gccgcggcgt tgtgacaatt
      241 taccgaacaa ctccgcggcc gggaagccga tctcggcttg aacgaattgt taggtggcgg
      301 tacttgggtc gatatcaaag tgcatcactt cttcccgtat gcccaacttt gtatagagag
      361 ccactgcggg atcgtcaccg taatctgctt gcacgtagat cacataagca ccaagcgcgt
      421 tggcctcatg cttgaggaga ttgatgagcg cggtggcaat gccctgcctc cggtgctcgc
      481 cggagactgc gagatcatag atatagatct cactacgcgg ctgctcaaac ctgggcagaa
      541 cgtaagccgc gagagcgcca acaaccgctt cttggtcgaa ggcagcaagc gcgatgaatg
      601 tcttactacg gagcaagttc ccgaggtaat cggagtccgg ctgatgttgg gagtaggtgg
      661 ctacgtctcc gaactcacga ccgaaaagat caagagcagc ccgcatggat ttgacttggt
      721 cagggccgag cctacatgtg cgaatgatgc ccatacttga gccacctaac tttgttttag
      781 ggcgactgcc ctgctgcgta acatcgttgc tgctgcgtaa catcgttgct gctccataac
      841 atcaaacatc gacccacggc gtaacgcgct tgctgcttgg atgcccgagg catagactgt
      901 acaaaaaaac agtcataaca agccatgaaa accgccactg cgccgttacc accgctgcgt
      961 tcggtcaagg ttctggacca gttgcgtgag cgcatacgct acttgcatta cagtttacga
     1021 accgaacagg cttatgtcaa ctgggttcgt gccttcatcc gtttccacgg tgtgcggcat
     1081 gcctgcagcg atctacacta gcactatcag cgttattaag caccggtgga gtgacgacct
     1141 tcagcacgtt cgtactgttc aacgatggtg tagtcttcgt tgtgggaggt gatgtccagt
     1201 ttgatgtcgg ttttgtaagc acccggcagc tgaaccggtt ttttagccat gtaggtggtt
     1261 ttaacttcag cgtcgtagtg accaccgtct ttcagtttca gacgcatttt gatttcacct
     1321 ttcagagcac cgtcttccgg gtacatacgt tcggtggaag cttcccaacc catggttttt
     1381 ttctgcataa ccggaccgtc ggacgggaag ttggtaccac gcagtttaac tttgtagatg
     1441 aactcaccgt cttgcaggga ggagtcctgg gtaacggtaa caacaccacc gtcttcgaag
     1501 ttcataacac gttcccattt gaaaccttcc gggaaggaca gtttcaggta gtccgggatg
     1561 tcagccgggt gtttaacgta agctttggaa ccgtactgga actgcgggga caggatgtcc
     1621 caagcgaacg gcagcggacc acctttggta actttcagtt tagcggtctg ggtaccttcg
     1681 tacggacgac cttcaccttc accttcgatt tcgaactcgt gaccgttaac ggaaccttcc
     1741 atacgaactt tgaaacgcat gaactctttg ataacgtctt cggaggaagc catctagtat
     1801 ttctcctctt tctctagtag ctagcacaat acctaggact gagctagccg tcaagcatgc
     1861 cgcgcccggg gagcccaagg gcacgccctg gcacctctag aggatccccg ggtaccgagc
     1921 tcgaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     1981 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     2041 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgata agctagcttc
     2101 acgctgccgc aagcactcag ggcgcaaggg ctgctaaagg aagcggaaca cgtagaaagc
     2161 cagtccgcag aaacggtgct gaccccggat gaatgtcagc tactgggcta tctggacaag
     2221 ggaaaacgca agcgcaaaga gaaagcaggt agcttgcagt gggcttacat ggcgatagct
     2281 agactgggcg gttttatgga cagcaagcga accggaattg ccagctgggg cgccctctgg
     2341 taaggttggg aagccctgca aagtaaactg gatggctttc ttgccgccaa ggatctgatg
     2401 gcgcagggga tcaagatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca
     2461 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     2521 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     2581 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactcc aagacgaggc
     2641 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
     2701 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     2761 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     2821 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     2881 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     2941 gctcgcgcca gccgaactgt tcgccaggct caaggcgcgg atgcccgacg gcgaggatct
     3001 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     3061 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
     3121 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
     3181 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
     3241 ctgagcggga ctctggggtt cgctagagga tcgatccttt ttaacccatc acatatacct
     3301 gccgttcact attatttagt gaaatgagat attatgatat tttctgaatt gtgattaaaa
     3361 aggcaacttt atgcccatgc aacagaaact ataaaaaata cagagaatga aaagaaacag
     3421 atagattttt tagttcttta ggcccgtagt ctgcaaatcc ttttatgatt ttctatcaaa
     3481 caaaagagga aaatagacca gttgcaatcc aaacgagagt ctaatagaat gaggtcgaaa
     3541 agtaaatcgc gcgggtttgt tactgataaa gcaggcaaga cctaaaatgt gtaaagggca
     3601 aagtgtatac tttggcgtca ccccttacat attttaggtc tttttttatt gtgcgtaact
     3661 aacttgccat cttcaaacag gagggctgga agaagcagac cgctaacaca gtacataaaa
     3721 aaggagacat gaacgatgaa catcaaaaag tttgcaaaac aagcaacagt attaaccttt
     3781 actaccgcac tgctggcagg aggcgcaact caagcgtttg cgaaagaaac gaaccaaaag
     3841 ccatataagg aaacatacgg catttcccat attacacgcc atgatatgct gcaaatccct
     3901 gaacagcaaa aaaatgaaaa atatcaagtt tctgaatttg attcgtccac aattaaaaat
     3961 atctcttctg caaaaggcct ggacgtttgg gacagctggc cattacaaaa cgctgacggc
     4021 actgtcgcaa actatcacgg ctaccacatc gtctttgcat tagccggaga tcctaaaaat
     4081 gcggatgaca catcgattta catgttctat caaaaagtcg gcgaaacttc tattgacagc
     4141 tggaaaaacg ctggccgcgt ctttaaagac agcgacaaat tcgatgcaaa tgattctatc
     4201 ctaaaagacc aaacacaaga atggtcaggt tcagccacat ttacatctga cggaaaaatc
     4261 cgtttattct acactgattt ctccggtaaa cattacggca aacaaacact gacaactgca
     4321 caagttaacg tatcagcatc agacagctct ttgaacatca acggtgtaga ggattataaa
     4381 tcaatctttg acggtgacgg aaaaacgtat caaaatgtac agcagttcat cgatgaaggc
     4441 aactacagct caggcgacaa ccatacgctg agagatcctc actacgtaga agataaaggc
     4501 cacaaatact tagtatttga agcaaacact ggaactgaag atggctacca aggcgaagaa
     4561 tctttattta acaaagcata ctatggcaaa agcacatcat tcttccgtca agaaagtcaa
     4621 aaacttctgc aaagcgataa aaaacgcacg gctgagttag caaacggcgc tctcggtatg
     4681 attgagctaa acgatgatta cacactgaaa aaagtgatga aaccgctgat tgcatctaac
     4741 acagtaacag atgaaattga acgcgcgaac gtctttaaaa tgaacggcaa atggtacctg
     4801 ttcactgact cccgcggatc aaaaatgacg attgacggca ttacgtctaa cgatatttac
     4861 atgcttggtt atgtttctaa ttctttaact ggcccataca agccgctgaa caaaactggc
     4921 cttgtgttaa aaatggatct tgatcctaac gatgtaacct ttacttactc acacttcgct
     4981 gtacctcaag cgaaaggaaa caatgtcgtg attacaagct atatgacaaa cagaggattc
     5041 tacgcagaca aacaatcaac gtttgcgccg agcttcctgc tgaacatcaa aggcaagaaa
     5101 acatctgttg tcaaagacag catccttgaa caaggacaat taacagttaa caaataaaaa
     5161 cgcaaaagaa aatgccgatg ggtaccgagc gaaatgaccg accaagcgac gcccaacctg
     5221 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     5281 ttccgggacg ccctcgcgga cgtgctcata gtccacgacg cccgtgattt tgtagccctg
     5341 gccgacggcc agcaggtagg ccgacaggct catgccggcc gccgccgcct tttcctcaat
     5401 cgctcttcgt tcgtctggaa ggcagtacac cttgataggt gggctgccct tcctggttgg
     5461 cttggtttca tcagccatcc gcttgccctc atctgttacg ccggcggtag ccggccagcc
     5521 tcgcagagca ggattcccgt tgagcaccgc caggtgcgaa taagggacag tgaagaagga
     5581 acacccgctc gcgggtgggc ctacttcacc tatcctgccc ggctgacgcc gttggataca
     5641 ccaaggaaag tctacacgaa ccctttggca aaatcctgta tatcgtgcga aaaaggatgg
     5701 atataccgaa aaaatcgcta taatgacccc gaagcagggt tatgcagcgg aaaagcgctg
     5761 cttccctgct gttttgtgga atatctaccg actggaaaca ggcaaatgca ggaaattact
     5821 gaactgaggg gacaggcgag agacgatgcc aaagagctcc tgaaaatctc gataactcaa
     5881 aaaatacgcc cggtagtgat cttatttcat tatggtgaaa gttggaacct cttacgtgcc
     5941 gatcaacgtc tcattttcgc caaaagttgg cccagggctt cccggtatca acagggacac
     6001 caggatttat ttattctgcg aagtgatctt ccgtcacagg tatttattcg gcgcaaagtg
     6061 cgtcgggtga tgctgccaac ttactgattt agtgtatgat ggtgtttttg aggtgctcca
     6121 gtggcttctg tttctatcag ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag
     6181 tgatcttatt tcattatggt gaaagttgga acctcttacg tgccgatcaa cgtctcattt
     6241 tcgccaaaag ttggcccagg gcttcccggt atcaacaggg acaccaggat ttatttattc
     6301 tgcgaagtga tcttccgtca caggtattta ttcggcgcaa agtgcgtcgg gtgatgctgc
     6361 caacttactg atttagtgta tgatggtgtt tttgaggtgc tccagtggct tctgtttcta
     6421 tcagggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     6481 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     6541 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     6601 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     6661 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     6721 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     6781 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     6841 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     6901 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     6961 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     7021 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     7081 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     7141 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     7201 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     7261 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     7321 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
     7381 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     7441 agcgggcagt gagcgcaacg caattaatgt gagttagctc a
//
