LOCUS       sequence-215131-        5782 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Recombination-mediated cassette exchange with target genomic loci.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5782)
  AUTHORS   Urtecho G, Tripp AD, Insigne K, Kim H, Kosuri S
  TITLE     Systematic Dissection of Sequence Elements Controlling sigma70 
            Promoters Using a Genomically-Encoded Multiplexed Reporter Assay in 
            E. coli.
  JOURNAL   Biochemistry. 2018 Feb 1. doi: 10.1021/acs.biochem.7b01069.
  PUBMED    29388765
REFERENCE   2  (bases 1 to 5782)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Biochemistry. 2018 Feb 1. doi: 10.1021/acs.biochem.7b01069."
COMMENT     Sequence Label: pLibacceptorV2
FEATURES             Location/Qualifiers
     source          1..5782
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(600..633)
                     /label=lox66
                     /bound_moiety="Cre recombinase"
                     /note="Right element (RE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1025..1819
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             1025..1819
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1079..1098)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1689..1708
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             complement(2166..3116)
                     /codon_start=1
                     /gene="rep101 encoding an A56V mutation"
                     /product="temperature-sensitive version of the RepA protein
                     needed for replication with the pSC101 origin (Armstrong et
                     al., 1984)"
                     /label=Rep101(Ts)
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      3164..3386
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             join(5179..5782,1..428)
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="MANLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
ORIGIN
        1 cgctggttag caccgcaggt gtagagaagg cacttagcct gggggtaact aaactggtcg
       61 agcgatggat ttccgtctct ggtgtagctg atgatccgaa taactacctg ttttgccggg
      121 tcagaaaaaa tggtgttgcc gcgccatctg ccaccagcca gctatcaact cgcgccctgg
      181 aagggatttt tgaagcaact catcgattga tttacggcgc taaggatgac tctggtcaga
      241 gatacctggc ctggtctgga cacagtgccc gtgtcggagc cgcgcgagat atggcccgcg
      301 ctggagtttc aataccggag atcatgcaag ctggtggctg gaccaatgta aatattgtca
      361 tgaactatat ccgtaacctg gatagtgaaa caggggcaat ggtgcgcctg ctggaagatg
      421 gcgattaact agtggggaag cttatcgatg ataagctgtc aaacatgaga attacaactt
      481 atatcgtatg gggctgactt caggtgctac atttgaagag ataaattgca ctgaaatcta
      541 gaaatatttt atctgattcc tcatcagtgc caacatagta agccagtaga attccgcgta
      601 taacttcgta tagcatacat tatacgaacg gtagacgaac aataaggcct cccaaatcgg
      661 ggggcctttt ttattgataa caaaactcgg taccaaattc cagaaaagag acgctgaaaa
      721 gcgtcttttt tcgttttggt ccagtgccat gttatccctg aagtcgacct agctcgatcg
      781 atggctatcc ctgcaggcat cgggtgggat ttagctacag gtatgcgtag gagtcaagtc
      841 agtttcacct gttttacgta aaaacccgct tcggcgggtt tttacttttg gtccggcaat
      901 taaaaaagcg gctaaccacg ccgctttttt tacgtctgca aaaaagagta ttgacttcgc
      961 atctttttgt acccataatt atttcattac tagaaagatc ttttaagaag gagatatata
     1021 ctagatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     1081 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     1141 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     1201 actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     1261 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     1321 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     1381 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     1441 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     1501 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc
     1561 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     1621 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     1681 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     1741 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     1801 tcttgacgag ttcttctgat gatactagag atcgagaagg acacggttaa tactaggcct
     1861 gctggctggt aatcgccagc aggccttttt atttggggga gagggaagtc atgaaaaaac
     1921 taacctttga aattcgatct ccaccacatc agctctgaag caacgtaaaa aaacccgccc
     1981 cggcgggttt ttttataccc gtagtatccc cacttatcta caatagctgt taccgttcgt
     2041 atatggtttc ttatacgaag ttatatatgg acagttttcc ctttgatatg taacggtgaa
     2101 cagttgttct acttttgttt gttagtcttg atgcttcact gatagataca agagccataa
     2161 gaacctcaga tccttccgta tttagccagt atgttctcta gtgtggttcg ttgtttttgc
     2221 gtgagccatg agaacgaacc attgagatca tacttacttt gcatgtcact caaaaatttt
     2281 gcctcaaaac tggtgagctg aatttttgca gttaaagcat cgtgtagtgt ttttcttagt
     2341 ccgttatgta ggtaggaatc tgatgtaatg gttgttggta ttttgtcacc attcattttt
     2401 atctggttgt tctcaagttc ggttacgaga tccatttgtc tatctagttc aacttggaaa
     2461 atcaacgtat cagtcgggcg gcctcgctta tcaaccacca atttcatatt gctgtaagtg
     2521 tttaaatctt tacttattgg tttcaaaacc cattggttaa gccttttaaa ctcatggtag
     2581 ttattttcaa gcattaacat gaacttaaat tcatcaaggc taatctctat atttgccttg
     2641 tgagttttct tttgtgttag ttcttttaat aaccactcat aaatcctcat agagtatttg
     2701 ttttcaaaag acttaacatg ttccagatta tattttatga atttttttaa ctggaaaaga
     2761 taaggcaata tctcttcact aaaaactaat tctaattttt cgcttgagaa cttggcatag
     2821 tttgtccact ggaaaatctc aaagccttta accaaaggat tcctgatttc cacagttctc
     2881 gtcatcagct ctctggttgc tttagctaat acaccataag cattttccct actgatgttc
     2941 atcatctgaa cgtattggtt ataagtgaac gataccgtcc gttctttcct tgtagggttt
     3001 tcaatcgtgg ggttgagtag tgccacacag cataaaatta gcttggtttc atgctccgtt
     3061 aagtcatagc gactaatcgc tagttcattt gctttgaaaa caactaattc agacatacat
     3121 ctcaattggt ctaggtgatt ttaatcacta taccaattga gatgggctag tcaatgataa
     3181 ttactagtcc ttttcctttg agttgtgggt atctgtaaat tctgctagac ctttgctgga
     3241 aaacttgtaa attctgctag accctctgta aattccgcta gacctttgtg tgtttttttt
     3301 gtttatattc aagtggttat aatttataga ataaagaaag aataaaaaaa gataaaaaga
     3361 atagatccca gccctgtgta taactcacta ctttagtcag ttccgcagta ttacaaaagg
     3421 atgtcgcaaa cgctgtttgc tcctctacaa aacagacctt aaaaccctaa aggcttaagt
     3481 agcaccctcg caagctcggt tgcggccgca atcgggcaaa tcgctgaata ttccttttgt
     3541 ctccgaccat caggcacctg agtcgctgtc tttttcgtga cattcagttc gctgcgctca
     3601 cggctctggc agtgaatggg ggtaaatggc actacaggcg ccttttatgg attcatgcaa
     3661 ggaaactacc cataatacaa gaaaagcccg tcacgggctt ctcagggcgt tttatggcgg
     3721 gtctgctatg tggtgctatc tgactttttg ctgttcagca gttcctgccc tctgattttc
     3781 cagtctgacc acttcggatt atcccgtgac aggtcattca gactggctaa tgcacccagt
     3841 aaggcagcgg tatcatcaac tttttttaag gcagttattg gtgcccttaa acgcctggtt
     3901 gctacgcctg aataagtgat aataagcgga tgaatggcag aaattcgaat agttacggct
     3961 tatgacatct ttgtggacac atcattcact ttttattcac atccggccct gaactcgcta
     4021 ggacttgccc cggtgcattt tttaaatacc cgcgaaaaat agagctgatc gtcaaatcca
     4081 acattgcgcc caacggtcgc tatcggcatt cgcgtagtgc taagcagaag tttcgcctgg
     4141 ctgatacgct gatcttcgcg ccagctcaat acgctaatgc ctaactgctg gcggaacaga
     4201 tgtgataacc gggagggcga caggcagaca tgctgggcga cgctggcgat atcaaaatgg
     4261 ctgtccgcca gatggtcgct gatatactgg caggcatcgc gcacacggct atccatcggc
     4321 gggtgcaacg actcattaat taccgccata cgtctgagca acaactgctc cagcagattg
     4381 atcgccagta gctcagaata gcgaccttcc ccttgcccgg cgctgatgat ctgcccgaac
     4441 agttcgctga aatgcggctg gcgcgcctcg tccgggcgga aaaatcctgt ctgggcaaag
     4501 attgtcggcc aggtcagcca ctcctgccag taggcgcgag gccggaaata aacccactgg
     4561 tgataccact cgctggcgtc cggatgccgt ccatagtgat gaatctcgcc cggcggaaac
     4621 aataatatat cgccaggccg acagacaaac tgctcgccat tattattaat gacgccctct
     4681 ccgcggatgg tcaggttaag aatatatccc ttcatgccca acggacgatc gataaaaaaa
     4741 tccagatatc cattcgcttc gatcggcgtc agcccggcga ccagatgggc attaaatgaa
     4801 tatcccggca atagcggatc attttgcgtt tcagccatga tttctctacc ccccgatgtt
     4861 cagagaagaa acaaattgtc catatcgacc aggacgacag agcttccgtc tccgcaagac
     4921 tttgcgcttg atgaaagcac gtatcaaccc cgcttgtgaa aagcgctttg taacaaaagc
     4981 gtacagttca ggcgataaaa ttaagtaaca gaagtgtcta taactatggc tggaatgtcc
     5041 acattgaata tttgcacagc gtcacacttt gcaaagcatt agcatttttg tccataagat
     5101 tagcggatcc tgcctgacgg tttttgccgc gactctctat aatttctcca tacctgtttt
     5161 tctggatgga gtaagaccat ggccaattta ctgaccgtac accaaaattt gcctgcatta
     5221 ccggtcgatg caacgagtga tgaggttcgc aagaacctga tggacatgtt cagggatcgc
     5281 caggcgtttt ctgagcatac ctggaaaatg cttctgtccg tttgccggtc gtgggcggca
     5341 tggtgcaagt tgaataaccg gaaatggttt cccgcagaac ctgaagatgt tcgcgattat
     5401 cttctatatc ttcaggcgcg cggtctggca gtaaaaacta tccagcaaca tttgggccag
     5461 ctaaacatgc ttcatcgtcg gtccgggctg ccacgaccaa gtgacagcaa tgctgtttca
     5521 ctggttatgc ggcggatccg aaaagaaaac gttgatgccg gtgaacgtgc aaaacaggct
     5581 ctagcgttcg aacgcactga tttcgaccag gttcgttcac tcatggaaaa tagcgatcgc
     5641 tgccaggata tacgtaatct ggcatttctg gggattgctt ataacaccct gttacgtata
     5701 gccgaaattg ccaggatcag ggttaaagat atctcacgta ctgacggtgg gagaatgtta
     5761 atccatattg gcagaacgaa aa
//
