LOCUS       sequence-467185-        7693 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Double-stranded recombineering in Staphylococcus aureus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7693)
  AUTHORS   Filsinger GT, Mychack A, Lyerly E, Henriksen C, Bartlett TM, 
            Kuchwara H, Eitzinger S, Bernhardt TG, Walker S, Church GM, Wannier 
            TM
  TITLE     A diverse single-stranded DNA-annealing protein library enables 
            efficient genome editing across bacterial phyla.
  JOURNAL   Proc Natl Acad Sci U S A. 2025 Apr 29;122(17):e2414342122. doi: 
            10.1073/pnas.2414342122. Epub 2025 Apr 21.
  PUBMED    40258142
REFERENCE   2  (bases 1 to 7693)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2025-04-29"; volume: "122(17):e2414342122. 
            doi"; pages: " 10.1073/pnas.2414342122. Epub 2025 Apr 21"
COMMENT     Sequence Label: pORTMAGE-Sa2
FEATURES             Location/Qualifiers
     source          1..7693
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     220..239
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     473..490
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    607..628
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(643..648,649..666,667..673)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    681..697
                     /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     686..708
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     705..721
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     705..721
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3405..3419
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             complement(3731..4357)
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     4262..4281
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     protein_bind    4464..4482
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     primer_bind     6142..6164
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6202..6220)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6288..6392
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6393..6461,6462..7253)
                     /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"
     primer_bind     complement(6611..6630)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7424..7693,1..319)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
       61 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
      121 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
      181 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
      241 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
      301 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
      361 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
      421 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
      481 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
      541 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcggaca gtgagcgcaa
      601 cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc
      661 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga
      721 ccatgattac gccaagcttc tgtaggtttt taggcataaa actatatgat ttacccctaa
      781 atctttaaaa tgccccttaa aattcaaaat aaaggcattt aaaatttaaa tatttcttgt
      841 gataaagttt gttaaaaagg agtggtttta tgactgttat gtggttatcg attataggta
      901 tgtggttttg tattggaatg gcattttttg ctatcaaggt tattaaaaat aaaaattaga
      961 ccacgcattt atgccgagaa aatttattgt gcgttgagaa gaacccttaa ctaaacttgc
     1021 agacgaatgt cggcatagcg tgagctatta agccgaccat tcgacaagtt ttgggattgt
     1081 taagggttcc gaggctcaac gtcaataaag caattggaat aaagaagcga aaaaggagaa
     1141 gtcggttcag aaaaagaagg atatggatct ggagctgtaa tataaaaacc ttcttcaact
     1201 aacggggcag gttagtgaca ttagaaaacc gactgtaaaa agtacagtcg gcattatctc
     1261 atattataaa agccagtcat taggcctatc tgacaattcc tgaatagagt tcataaacaa
     1321 tcctgcatga taaccatcac aaacagaatg atgtacctgt aaagatagcg gtaaatatat
     1381 tgaattacct ttattaatga attttcctgc tgtaataatg ggtagaaggt aattactatt
     1441 attattgata tttaagttaa acccagtaaa tgaagtccat ggaataatag aaagagaaaa
     1501 agcattttca ggtataggtg ttttgggaaa caatttcccc gaaccattat atttctctac
     1561 atcagaaagg tataaatcat aaaactcttt gaagtcattc tttacaggag tccaaatacc
     1621 agagaatgtt ttagatacac catcaaaaat tgtataaagt ggctctaact tatcccaata
     1681 acctaactct ccgtcgctat tgtaaccagt tctaaaagct gtatttgagt ttatcaccct
     1741 tgtcactaag aaaataaatg cagggtaaaa tttatatcct tcttgtttta tgtttcggta
     1801 taaaacacta atatcaattt ctgtggttat actaaaagtc gtttgttggt tcaaataatg
     1861 attaaatatc tcttttctct tccaattgtc taaatcaatt ttattaaagt tcatttgata
     1921 tgcctcctaa atttttatct aaagtgaatt taggaggctt acttgtctgc tttcttcatt
     1981 agaatcaatc cttttttaaa agtcaatatt actgtaacat aaatatatat tttaaaaata
     2041 tcccacttta tccaattttc gtttgttgaa ctaatgggtg ctttagttga agaataaaag
     2101 accacattaa aaaatgtggt cttttgtgtt tttttaaagg atttgagcgt agcgaaaaat
     2161 ccttttcttt cttatcttga taataagggt aactattgcc ggcgaggcta gttaccctta
     2221 agttattggt atgactggtt ttaagcgcaa aaaaagttgc tttttcgtac ctattaatgt
     2281 atcgttttaa atgaatagta aaaaacatac atagaaaggg gaaaaagcaa ctttttttat
     2341 tgtcatagtt tgtgaaaact aagttgtttt tatgtgttat aacatggaaa agtatactga
     2401 gaaaaaacaa agaaatcaag tatttcagaa atttattaaa cgtcatattg gagagaatca
     2461 aatggattta gttgaagatt gcaatacatt tctgtctttt gtagctgata aaactttaga
     2521 aaaacagaaa ttatataaag ctaattcttg taaaaatcga ttttgtcctg tctgtgcttg
     2581 gagaaaagct agaaaagatg cattgggttt atctttgatg atgcaatata ttaagcagca
     2641 agagaaaaag gagtttatct ttttaacttt gactacacct aatgtaatga gtgatgaatt
     2701 agaaaatgaa ataaaacgtt ataataattc ttttagaaaa cttataaaga gaaaaaaagt
     2761 aggtagtgtt ataaagggat atgttcgtaa gttagagatt acatataata aaaaaagaga
     2821 tgattataat cctcattttc atgtgttaat tgcagtaaat aaatcgtatt tcacagataa
     2881 aagatattat attagccaac aagaatggtt agatttatgg cgtgatgtaa cgggcatttc
     2941 agaaataaca caagttcaag ttcaaaaaat aagacaaaat aataataaag aattatatga
     3001 aatggctaag tattctggta aagatagtga ttatttaata aatcaaaaag tctttgatgc
     3061 attttataaa tcacttaaag gtaaacaggt attagtttat tcaggattat ttaaagaggc
     3121 taaaaagaaa ttaaaaaatg gggatttaga ttacttaaaa gaaattgatc caaccgaata
     3181 tatctatcaa attttttata tttggaaaca aaaagagtat ttagctagtg aactttatga
     3241 cttaacagaa caagaaaaaa gagaaattaa tcacaaaatg atagacgaaa tcgaggaaga
     3301 acaataacaa aatataagtg ctaacagctg acctcccgat aacaccatgt agttattggg
     3361 aggtcagctg ttgaattatg cacgagtatt ttaaaagtta ttgtgatgac gacgataaac
     3421 gattatcaaa agtataatgt taaaatgctt tattatacta acgttatata aacattatac
     3481 tttcgttata caaattttaa ccctgttagg aactataaaa aatcatgaaa attttaattt
     3541 gcatgtaact gggcagtgtc ttaaaaaatc gacactgaat ttgctcaaat ttttgtttgt
     3601 agaattagaa tatatttatt tggctcatat ttgcttttta aaagcttgca tgcctgcagg
     3661 tcgacggtat cgataactcg acatcttggt taccgtgaag ttaccatcac ggaaaaaggt
     3721 tatgctgctt ttaagaccca ctttcacatt taagttgttt ttctaatccg catatgatca
     3781 attcaaggcc gaataagaag gctggctctg caccttggtg atcaaataat tcgatagctt
     3841 gtcgtaataa tggcggcata ctatcagtag taggtgtttc cctttcttct ttagcgactt
     3901 gatgctcttg atcttccaat acgcaaccta aagtaaaatg ccccacagcg ctgagtgcat
     3961 ataatgcatt ctctagtgaa aaaccttgtt ggcataaaaa ggctaattga ttttcgagag
     4021 tttcatactg tttttctgta ggccgtgtac ctaaatgtac ttttgctcca tcgcgatgac
     4081 ttagtaaagc acatctaaaa cttttagcgt tattacgtaa aaaatcttgc cagctttccc
     4141 cttctaaagg gcaaaagtga gtatggtgcc tatctaacat ctcaatggct aaggcgtcga
     4201 gcaaagcccg cttatttttt acatgccaat acaatgtagg ctgctctaca cctagcttct
     4261 gggcgagttt acgggttgtt aaaccttcga ttccgacctc attaagcagc tctaatgcgc
     4321 tgttaatcac tttactttta tctaatctag acatcattaa ttcctccttt ttgttgacat
     4381 tatatcattg atagagttat ttgtcaaact agttttttat ttggatcccc tcgagttcat
     4441 gaaaaactaa aaaaaatatt gacactctat cattgataga gtataattaa aataagctct
     4501 ctatcattga tagagtaata aacaccaaag gaggcctttt atggctgaaa acgaaaaaca
     4561 agctttatta caagaagaaa acaaatcaga aaacgttgtt tcaactgtta aacgtactgc
     4621 tttagctact aacccattct cagatactga tcaattcaac aacatcttca aaatggctca
     4681 attaatctca caatcagata tgatcccagc tacttacaaa ggtaaaccaa tgaactgtgt
     4741 tatcgcttta gaacaagcta accgtatggg tgtttcacca ttaatggtta tgcaaaactt
     4801 atacgttgtt aaaggtgttc catcatggtc aggtcaaggt tgtatgatga tcatccaagg
     4861 ttgtggtaaa ttccgtgatg ttgattacgt ttactcaggt gaaaaaggta ctgattcacg
     4921 ttcatgtaaa gttgttgcta ctcgtatctc agatggtaaa cgtatcgaag gtactgaaat
     4981 cactatgcaa atggttaaat cagaaggttg gatctcaaac actaaatgga aaaacatgcc
     5041 agaacaaatg ttaggttacc gtgctgctac tttcttcgct cgtatgtact gtccaaacga
     5101 attaaacggt ttcgctactg aaggtgaagc tgaagatatg aaccacaaac cacaacgtat
     5161 cgaagctatc aacgttttag gtgatactgc tcacgaataa aagccgcaga gcagaaggtg
     5221 gcagcatgaa caaaggcttc attctttctc aagagaacta ttacttgccg gagactaatc
     5281 aacttttctg ctctgcctct caatacaagc aagccattgg gtgtattgga cgccctgggt
     5341 gcgaggctac gatgcttgcc caattgcgtg gtgactggga gtttgagacc tctattgaca
     5401 tgctgatcgg gtcgtatgta gatgcccgct acgagggtac aatcgagcag ttccaggagg
     5461 agcacccgga attgttcgtg agtcgtggtg agcgtaaagg tcagcttaaa gctgaattca
     5521 tcaaagcaga acaaatctat cgtcgttgcg agaaggatac gctgttctcc gcctatatgt
     5581 ccggtgaaaa gcaagtaatc atgacgggag agattgccgg agtgccattt aaaattaaga
     5641 tggacagttt acaccgcgga aagtgcatcg tggacttaaa ggtgatgcaa gacttgtcta
     5701 aacagttttg ggtacgcgat tacggccata tggacttcat cacatattgg ggatatgata
     5761 ttcaacttgc catttaccaa gagatttatt accaaaatac tggggaacgc ttaccaacgt
     5821 atatctgtgc tgcagataag gggaaggtaa cagatatcgc ggtcatttat gtagacgata
     5881 tgcgtttacg tgagtgtctg gaagcagtca aggccaacgc acctaaaatc caaatgctga
     5941 aatctggcga gatcgaaccg attcgctgcg agttgtgcga ctactgcaag ttcaccaagg
     6001 ttttggataa acccattcat ttctccgaat tgacgggcga cgtgtaatta agccagcccc
     6061 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     6121 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     6181 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     6241 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     6301 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     6361 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     6421 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     6481 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     6541 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     6601 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     6661 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     6721 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     6781 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     6841 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     6901 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     6961 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     7021 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     7081 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     7141 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     7201 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     7261 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     7321 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     7381 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     7441 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     7501 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     7561 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     7621 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     7681 cgtgtcttac cgg
//
