LOCUS       sequence-467186-        8157 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Single-stranded and double-stranded recombineering in 
            Corynebacterium glutamicum.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8157)
  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 8157)
  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-Cg1
FEATURES             Location/Qualifiers
     source          1..8157
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             204..998
                     /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(258..277)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     868..887
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      1098..1686
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1587..1606
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1840..1857
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(2000..3082)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     3044..3063
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3083..3160)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(3483..3488,3489..3504,3505..3511)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    3519..3535
                     /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)."
     CDS             4951..4968
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      5098..5184
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5277..5304
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 aggtagcttg cagtgggctt acatggcgat agctagactg ggcggtttta tggacagcaa
       61 gcgaaccgga attgccagct ggggcgccct ctggtaaggt tgggaagccc tgcagagtaa
      121 actggatggc tttcttgccg ccaaggatct gatggcgcag gggatcaagg tctgatcaag
      181 agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg
      241 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
      301 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
      361 tgtccggtgc cctgaatgaa ctccaagacg aggcagcgcg gctatcgtgg ctggccacga
      421 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
      481 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
      541 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
      601 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
      661 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
      721 ggctcaaggc gcggatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
      781 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
      841 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
      901 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
      961 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgcgga
     1021 atcatgacca aaatccctta acgtgagttt tcgttccagc atgcgtcaga ccccgtagaa
     1081 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     1141 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     1201 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     1261 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     1321 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     1381 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     1441 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     1501 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     1561 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     1621 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     1681 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     1741 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     1801 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     1861 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     1921 ataaaagatc tctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
     1981 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     2041 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     2101 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     2161 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
     2221 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     2281 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     2341 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     2401 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     2461 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
     2521 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
     2581 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
     2641 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
     2701 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     2761 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     2821 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     2881 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     2941 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     3001 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     3061 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     3121 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
     3181 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc
     3241 gccgcaagga atggtgcatg atatggatcc atatatgcgg ccgcatattc tagaatccgg
     3301 agcttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
     3361 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
     3421 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
     3481 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttgc
     3541 tagcgaaagg attttttaca tatgaacgaa atcattaaaa aagaggccac tgagatcatt
     3601 acccctgtcg acgagaaaac tattatggaa taccttgaca cgacgggatt gaccaagtca
     3661 ttgctcccca aggagaaagc aatgtttgta aacatggctc ggctctacgg acttaatcct
     3721 ttcaaacgtg agatttactg cacggtgtat ggagaaggac aatatcgtca gtgctccatc
     3781 gttacgggat acgaagtata cctcaaacgt gcggaacgca ttggaaagct ggatggctgg
     3841 caagcacaaa ttaccggatc tttgcaagac ggcacgcttg ccgccactgt aacgatttgg
     3901 cgcaaagatt ggacgcatcc cttcacgcat actgcatttt atacggaatg tgtgcagacc
     3961 tcaaagaaaa cgggtgaacc gaatgcgatc tggcgcaaga tgccatcatt tatggtacgt
     4021 aaggtggcaa tcgcgcaagc gtttcggctc tgcttttcag acgaatttgg cggtatgccg
     4081 tacaccaacg atgaaatggg agtcgatgca ccgaaagaac gggatattac tcacgaggct
     4141 accgccacca ttgcggacga agccgaagct ccgtctgcgg aagttaaaaa tgagcccaag
     4201 cccgccgatg ttgtacaaca actggagacg ctcttgatga aatatgaatc ccaactttct
     4261 ggcaagcctt acgaactggc ggaggaagca ttgcggactg gtagcgacgc agaagtaatc
     4321 gctatgtatg aacgcgtagt atcttatctg aaacgcaagg gaatccaagt gggtaagtaa
     4381 aggagagcca gtgtgacgct tactaatgac ggacattaac catgttttgg ttatcggacg
     4441 gttgactcgt gactttggcg cggaccctcg tactttcttc tatacgacgg gtggtacggc
     4501 ttgcgctaaa gtgagcatcg ccgtgaatcg cagcgtaaag caatccgacg gtcaatggac
     4561 cgacgaggtt tccttctttg acgttaccat ttggggaaag actgctgaaa atctgaagcc
     4621 ataccttgtg aagggaaaac aaatcgccgt tgatggatat ttgaagcaag accgttggca
     4681 aaaagatggc caaaattttt ctaaagtcaa tatcgtcgct aattcagtcc aactcctcgg
     4741 cggaggtacc tctgctcctg aatcagcacc ggcccctcag aactatggtc gggtccagga
     4801 aacttatcgg gacaatccaa gccaagtgcc accacgtatg caaggaaatt acatgcaacc
     4861 tcagcaaccc gcgtatcaaa ccaccgcacc tcaacagcag tttggaggcg gagatgactt
     4921 ccccgaggac ctgcctttct aactaagctt caccaccacc accaccactg acctaggtgc
     4981 ctggcggcag tagcgcggtg gtcccacctg accccatgcc gaactcagaa gtgaaacgcc
     5041 gtagcgccga tggtagtgtg gggtctcccc atgcgagagt agggaactgc caggcatcaa
     5101 ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg
     5161 aacgctctcc tgagtaggac aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg
     5221 cccggaaggg tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa
     5281 ggccatcctg acggatggcc tttttactag tatatattaa ttaaatatgg tacctttaat
     5341 tccgctagat gacgtgcggc ttcgacctcc tgggcgtggc gcttgttggc gcgctcgcgg
     5401 ctggctgcgg cacgacacgc gtctgagcag tcaattgcgc gccgtcctcg tgggtcaggc
     5461 cggggtggga tcaggccacc gcagtaggcg cagctgatgc gatcctccac tactgcgcgt
     5521 cctcctggcg ctgccgagca cgcagctcgt cggccagctc ttcaaggtcg gccacaagcg
     5581 tttctaggtc gctcgcggca cttgcccagt cgcgtgatgc tggcgcgtct gtcgtatcga
     5641 gggcgcggaa aaatccgatc accgttttta aatcgacggc ggcatcgagt gcgtcggact
     5701 ccagcgcgac atcggagaga tccaccgctg atgcttcagg ccagttttgg tacttcgtcg
     5761 tgaaggtcat gacaccatta taacgaacgt tcgttaaaaa ttctagcccc aattctgata
     5821 atttcttccg gcactcctgc gaaaacctgc gagacttctt gcccagaaaa aacgccaagc
     5881 gcagcggtta ccgcactttt tttccaggtg atttcaccct gaccagcgaa gcggcacttt
     5941 agtgcatgag gtgtgcccct ggtttcccct ctttggaggg ttcaacccaa aaaagcacac
     6001 aagcaaaaat gaaaatcatc atgagcaagt tggtgcgaag cagcaacgcg caagctccaa
     6061 aaaggtctcc aggatctcga ggagattttt gagggggagg gagtcgagga agagccagag
     6121 cagaaggcgg ggaaccgttc tctgccgaca gcgtgagccc cccttaaaaa tcaggccggg
     6181 gaggaaccgg ggagggatca gagctaggag cgagacaccc taaagggggg gaaccgtttt
     6241 ctgctgacgg tgtttcgttt attagttttc agcccgtgga tagcggaggg tgagggcaag
     6301 tgagagccag agcaaggacg ggacccctaa aggggggaac cgttttctgc tgacggtgtt
     6361 tcgtttatta gttttcagcc cgtggacggc cgcgtttagc ttccattcca agtgcctttc
     6421 tgacttgttg gatgcgcctt tcactgacac ctagttcgcc tgcaagctca cgagtcgagg
     6481 gatcagcaac cgattgagaa cgggcatcca ggatcgcagt tttgacgcga agttcgagca
     6541 actcgcctgt catttctcgg cgtttgtttg cttccgctaa tcgctgtcgc gtctcctgcg
     6601 catacttact ttctgggtca gcccatctgc gtgcattcga tgtagctgcg ccccgtcgcc
     6661 ccatcgtcgc tagagctttc cgccctcggc tgctctgcgt ttccacccga cgagcaggga
     6721 cgactggctg gcctttagcc acgtagccgc gcacacgacg cgccatcgtc aggcgatcac
     6781 gcatggcggg aagatccggc tcccggccgt ctgcaccgac cgcctgggca acgttgtacg
     6841 ccacttcata cgcgtcgatg atcttggcat cttttaggcg ctcaccagca gctttgagct
     6901 ggtatcccac ggtcaacgcg tggcgaaacg cggtctcgtc gcgcgctcgc tctggatttg
     6961 tccagagcac tcgcacgccg tcgatcaggt cgccggacgc gtccagggcg ctcggcaggc
     7021 tcgcgtccaa aatcgctagt gccttggctt ctgcggtggc gcgttgtgcc gcttcaatgc
     7081 gggcgcgtcc gctggaaaag tcctgctcaa tgtacttttt cggcttctgt gatccggtca
     7141 tcgttcgagc aatctccatt aggtcggcca gccgatccac acgatcatgc tggcagtgcc
     7201 atttataggc tgtcggatcg tctgagacgt gcagcggcca ccggctcagc ctatgcgaaa
     7261 aagcctggtc agcgccgaaa acacgagtca tttcttccgt cgttgcagcc agcaggcgca
     7321 tatttgggct ggttttacct gctgcggcat acaccgggtc aatgagccag atgagctggc
     7381 atttcccgct cagcggattc acgccgatcc aagccggcgc tttttctagg cgtgcccatt
     7441 tctctaaaat cgcgtagacc tgcgggttta cgtgctcaat cttcccgccg gcctggtggc
     7501 tgggcacatc gatgtcaagc acgatcaccg cggcatgttg cgcgtgcgtc agcgcaacgt
     7561 actggcaccg cgtcagcgct tttgagccag cccggtagag ctttggttgg gtttcgccgg
     7621 tatccgggtt tttaatccag gcgctcgcga aatctcttgt cttgctgccc tggaagctct
     7681 cgcgtcccag gtgagcgagc agttcgcggc gatcttctgc cgtccagccg cgtgagccgc
     7741 agcgcatagc ttcggggtgg gtgtcgaaca gatcggcgga caatttccac gcgcttgctg
     7801 tgactgtgtc ctgcggatcg gctagagtca tgtcttgagt gctttctccc agctgatgac
     7861 tgggggttag ccgacgccct gtgagttccc gctcacgggg cgttcaactt tttcaggtat
     7921 ttgtgcagct tatcgtgttt tcttcgtaaa tgaacgctta actaccttgt taaacgtggc
     7981 aaataggcag gattgatggg gatctagctt cacgctgccg caagcactca gggcgcaagg
     8041 gctgctaaag gaagcggaac acgtagaaag ccagtccgca gaaacggtgc tgaccccgga
     8101 tgaatgtcag ctactgggct atctggacaa gggaaaacgc aagcgcaaag agaaagc
//
