LOCUS       sequence-397976-        3949 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 1 plasmid, barcoded ORI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3949)
  AUTHORS   Gilbert C, Brumwell SL, Crits-Christoph A, Kang SC, Martin-Moldes Z,
            Alsharif W, Esmurria A, Nguyen M-A, Lee HH, Ostrov N
  TITLE     A scalable framework for high-throughput identification of 
            functional origins of replication in non-model bacteria
  JOURNAL   bioRxiv 2023.05.19.541510
REFERENCE   2  (bases 1 to 3949)
  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: "bioRxiv 
            2023.05.19.541510"
COMMENT     Sequence Label: pGL1_2
FEATURES             Location/Qualifiers
     source          1..3949
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     593..612
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     677..696
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     806..822
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     806..822
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     terminator      823..869
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     983..1005
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     997..1014
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     998..1014
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1711..2715
                     /codon_start=1
                     /gene="repB"
                     /product="RepB replication protein"
                     /label=repB
                     /note="from Enterococcus faecalis plasmid pAM-alpha-1"
                     /db_xref="GI:22652809"
                     /protein_id="AAN03827.1"
                     /translation="MGVSFNIMCPNSSIYSDEKSRVLVDKTKSGKVRPWREKKIANVDY
                     FELLHILEFKKAERVKDCAEILEYKQNRETGERKLYRVWFCKSRLCPMCNWRRAMKHGI
                     QSQKVVAEVIKQKPTVRWLFLTLTVKNVYDGEELNKSLSDMAQGFRRMMQYKKINKNLV
                     GFMRATEVTINNKDNSYNQHMHVLVCVEPTYFKNTENYVNQKQWIQFWKKAMKLDYDPN
                     VKVQMIRPKNKYKSDIQSAIDETAKYPVKDTDFMTDDEEKNLKRLSDLEEGLHRKRLIS
                     YGGLLKEIHKKLNLDDTEEGDLIHTDDDEKADEDGFSIIAMWNWERKNYFIKE"
     primer_bind     complement(2907..2923)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2907..2923)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2920..2942)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(3095..3683)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3175..3194)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3819..3949,1..685))
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(join(3819..3949,1..685))
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
ORIGIN
        1 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
       61 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
      121 agaatggcaa aagcttatgc atttctttcc agacttgttc aacaggccag ccattacgct
      181 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
      241 gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
      301 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
      361 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
      421 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
      481 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
      541 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
      601 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctcgagcaag
      661 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca
      721 gttttattgt tcatgatgat atatttttat cttgtgcaat gtaacatcag agattttgag
      781 acacaacgtg gctttccccc gccgctctag aactagtgga tccaaataaa acgaaaggct
      841 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cgcattatac gagacgtcca
      901 ggttgggata cctgaaacaa aacccatcgt acggccaagg aagtctccaa taactgtgat
      961 ccaccacaag cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtcatgca
     1021 taatccgcac gcatctggaa taaggaagtg ccattccgcc tgaccttagc gtgcgaacag
     1081 cacgctattg ttgtatcatt actcgcatcc attctcaggc tgtctcgtct cgtctccgag
     1141 ctcttcaatg agtcagggtc acgcgtagga cgcgtcgagt agggtccctt taatcagatg
     1201 cgtcggttcc gcagccacac gccgtgtgaa gctggggagc taaaataagc gatttaaagc
     1261 gtgatgtgag tttaatctat gaaagcacta aggaattcct taaggaacgt acagacggct
     1321 taaaagcctt taaaaacgtt tttaaggggt ttgtagacaa ggtaaaggat aaaacagcac
     1381 aattccaaga aaaacacgat ttagaaccta aaaagaacga atttgaacta actcataacc
     1441 gagaggtaaa aaaagaacga agtcgagatc agggaatgag tttataaaat aaaaaaagca
     1501 cctgaaaagg tgtctttttt tgatggtttt gaacttgttc tttcttatct tgatacatat
     1561 agaaataacg tcatttttat tttagttgct gaaaggtgcg ttgaagtgtt ggtatgtatg
     1621 tgttttaaag tattgaaaac ccttaaaatt ggttgcacag aaaaacccca tctgttaaag
     1681 ttataagtga ctaaacaaat aactaaatag atgggggttt cttttaatat tatgtgtcct
     1741 aatagtagca tttattcaga tgaaaaatca agggttttag tggacaagac aaaaagtgga
     1801 aaagttagac catggagaga aaagaaaatc gctaatgttg attactttga acttctgcat
     1861 attcttgaat ttaaaaaggc tgaaagagta aaagattgtg ctgaaatatt agagtataaa
     1921 caaaatcgtg aaacaggcga aagaaagttg tatcgagtgt ggttttgtaa atccaggctt
     1981 tgtccaatgt gcaactggag gagagcaatg aaacatggca ttcagtcaca aaaggttgtt
     2041 gctgaagtta ttaaacaaaa gccaacagtt cgttggttgt ttctcacatt aacagttaaa
     2101 aatgtttatg atggcgaaga attaaataag agtttgtcag atatggctca aggatttcgc
     2161 cgaatgatgc aatataaaaa aattaataaa aatcttgttg gttttatgcg tgcaacggaa
     2221 gtgacaataa ataataaaga taattcttat aatcagcaca tgcatgtatt ggtatgtgtg
     2281 gaaccaactt attttaagaa tacagaaaac tacgtgaatc aaaaacaatg gattcaattt
     2341 tggaaaaagg caatgaaatt agactatgat ccaaatgtaa aagttcaaat gattcgaccg
     2401 aaaaataaat ataaatcgga tatacaatcg gcaattgacg aaactgcaaa atatcctgta
     2461 aaggatacgg attttatgac cgatgatgaa gaaaagaatt tgaaacgttt gtctgatttg
     2521 gaggaaggtt tacaccgtaa aaggttaatc tcctatggtg gtttgttaaa agaaatacat
     2581 aaaaaattaa accttgatga cacagaagaa ggcgatttga ttcatacaga tgatgacgaa
     2641 aaagccgatg aagatggatt ttctattatt gcaatgtgga attgggaacg gaaaaattat
     2701 tttattaaag agtagttcaa caaacgggcc agtttgttga agattagatg ctataattgc
     2761 gctagcatga agagcacccc aggatacata gattaccaca actccgagcc cttccaccta
     2821 gcgtgctaac cacgcacgct attgttgtat aggctaggtg gaggctcagt gatgataagt
     2881 ctgcgatggt ggatgcatgt gtcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     2941 ctcagagggc acaatcctat tccgcgctat ccgacaatct ccaagacatt aggtggagtt
     3001 cagttcggcg tatggcatat gtcgctggaa agaacatgtg agcaaaaggc cagcaaaagg
     3061 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     3121 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     3181 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     3241 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     3301 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     3361 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     3421 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     3481 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     3541 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     3601 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     3661 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     3721 tattcaacaa agccgccgtc ccgtcaagtc agcgtaaatg ggtagggggc ttcaaatcgt
     3781 ccgctctgcc agtgttacaa ccaattaaca aattctgatt agaaaaactc atcgagcatc
     3841 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt
     3901 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaa
//
