LOCUS       sequence-398480-        4651 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 4651)
  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 4651)
  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_35
FEATURES             Location/Qualifiers
     source          1..4651
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     89..108
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     173..192
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     302..318
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     302..318
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     terminator      319..365
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     479..501
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     493..510
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     494..510
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(786..1934)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     IVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     rep_origin      2327..2858
                     /label=oriV
                     /note="incP origin of replication"
     primer_bind     complement(3105..3121)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3105..3121)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3118..3140)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(3293..3881)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3373..3392)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4017..4651,1..181))
                     /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(4017..4651,1..181))
                     /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 cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat
       61 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag
      121 catccatgtt ggaatttaat cgcggcctcg agcaagacgt ttcccgttga atatggctca
      181 taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat gatgatatat
      241 ttttatcttg tgcaatgtaa catcagagat tttgagacac aacgtggctt tcccccgccg
      301 ctctagaact agtggatcca aataaaacga aaggctcagt cgaaagactg ggcctttcgt
      361 tttatctgtt gtttgtcgca ttatacgaga cgtccaggtt gggatacctg aaacaaaacc
      421 catcgtacgg ccaaggaagt ctccaataac tgtgatccac cacaagcgcc agggttttcc
      481 cagtcacgac gttgtaaaac gacggccagt catgcataat ccgcacgcat ctggaataag
      541 gaagtgccat tccgcctgac cttagcgtgc gaacagcacg ctattgttgt atcattactc
      601 gcatccattc tcaggctgtc tcgtctcgtc tccgagctct tcaatgagtc agggtcacgc
      661 gtaggacgcg tcgagtaggg ttagattccc ttgacttgcg tgttccgcag ccacacgccg
      721 tgtgaagctg gggaggcgat gcaggtggct gctgaacccc cagccggaac tgaccccaca
      781 aggccctagc gtttgcaatg caccaggtca tcattgaccc aggcgtgttc caccaggccg
      841 ctgcctcgca actcttcgca ggcttcgccg acctgctcgc gccacttctt cacgcgggtg
      901 gaatccgatc cgcacatgag gcggaaggtt tccagcttga gcgggtacgg ctcccggtgc
      961 gagctgaaat agtcgaacat ccgtcgggcc gtcggcgaca gcttgcggta cttctcccat
     1021 atgaatttcg tgtagtggtc gccagcaaac agcacgacga tttcctcgtc gatcaggacc
     1081 tggcaacggg acgttttctt gccacggtcc aggacgcgga agcggtgcag cagcgacacc
     1141 gattccaggt gcccaacgcg gtcggacgtg aagcccatcg ccgtcgcctg taggcgcgac
     1201 aggcattcct cggccttcgt gtaataccgg ccattgatcg accagcccag gtcctggcaa
     1261 agctcgtaga acgtgaaggt gatcggctcg ccgatagggg tgcgcttcgc gtactccaac
     1321 acctgctgcc acaccagttc gtcatcgtcg gcccgcagct cgacgccggt gtaggtgatc
     1381 ttcacgtcct tgttgacgtg gaaaatgacc ttgttttgca gcgcctcgcg cgggattttc
     1441 ttgttgcgcg tggtgaacag ggcagagcgg gccgtgtcgt ttggcatcgc tcgcatcgtg
     1501 tccggccacg gcgcaatatc gaacaaggaa agctgcattt ccttgatctg ctgcttcgtg
     1561 tgtttcagca acgcggcctg cttggcttcg ctgacctgtt ttgccaggtc ctcgccggcg
     1621 gtttttcgct tcttggtcgt catagttcct cgcgtgtcga tggtcatcga cttcgccaaa
     1681 cctgccgcct cctgttcgag acgacgcgaa cgctccacgg cggccgatgg cgcgggcagg
     1741 gcagggggag ccagttgcac gctgtcgcgc tcgatcttgg ccgtagcttg ctggactatc
     1801 gagccgacgg actggaaggt ttcgcggggc gcacgcatga cggtgcggct tgcgatggtt
     1861 tcggcatcct cggcggaaaa ccccgcgtcg atcagttctt gcctgtatgc cttccggtca
     1921 aacgtccgat tcattcaccc tccttgcggg attgccccgg aattaattcc ccggatcgat
     1981 ccgtcgatct tgatcccctg cgccatcaga tccttggcgg caagaaagcc atccagttta
     2041 ctttgcaggg cttcccaacc ttaccagagg gcgccccagc tggcaattcc ggttcgcttg
     2101 ctgtccataa aaccgcccag tctagctatc gccatgtaag cccactgcaa gctacctgct
     2161 ttctctttgc gcttgcgttt tcccttgtcc agatagccca gtagctgaca ttcatccggg
     2221 gtcagcaccg tttctgcgga ctggctttct acgtggctgc catttttggg gtgaggccgt
     2281 tcgcggccga ggggcgcagc ccctgggggg atgggaggcc cgcgttagcg ggccgggagg
     2341 gttcgagaag ggggggcacc ccccttcggc gtgcgcggtc acgcgcacag ggcgcagccc
     2401 tggttaaaaa caaggtttat aaatattggt ttaaaagcag gttaaaagac aggttagcgg
     2461 tggccgaaaa acgggcggaa acccttgcaa atgctggatt ttctgcctgt ggacagcccc
     2521 tcaaatgtca ataggtgcgc ccctcatctg tcagcactct gcccctcaag tgtcaaggat
     2581 cgcgcccctc atctgtcagt agtcgcgccc ctcaagtgtc aataccgcag ggcacttatc
     2641 cccaggcttg tccacatcat ctgtgggaaa ctcgcgtaaa atcaggcgtt ttcgccgatt
     2701 tgcgaggctg gccagctcca cgtcgccggc cgaaatcgag cctgcccctc atctgtcaac
     2761 gccgcgccgg gtgagtcggc ccctcaagtg tcaacgtccg cccctcatct gtcagtgagg
     2821 gccaagtttt ccgcgaggta tccacaacgc cggcggccct acatggctct gctgtagtga
     2881 gtgggttgcg ctccggcagc ggtcctgatc ccccgcagaa aaaaaggatc tcaagaagat
     2941 cctttgatct tttctaccgc tagcatgaag agcaccccag gatacataga ttaccacaac
     3001 tccgagccct tccacctagc gtgctaacca cgcacgctat tgttgtatag gctaggtgga
     3061 ggctcagtga tgataagtct gcgatggtgg atgcatgtgt catggtcata gctgtttcct
     3121 gtgtgaaatt gttatccgct cagagggcac aatcctattc cgcgctatcc gacaatctcc
     3181 aagacattag gtggagttca gttcggcgta tggcatatgt cgctggaaag aacatgtgag
     3241 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     3301 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     3361 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     3421 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     3481 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     3541 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     3601 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     3661 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     3721 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     3781 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     3841 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     3901 ctacggggtc tgacgctcta ttcaacaaag ccgccgtccc gtcaagtcag cgtaaatggg
     3961 tagggggctt caaatcgtcc gctctgccag tgttacaacc aattaacaaa ttctgattag
     4021 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca
     4081 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg
     4141 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt
     4201 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa
     4261 tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac aggccagcca
     4321 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc
     4381 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc
     4441 aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct
     4501 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca
     4561 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt
     4621 ctgaccatct catctgtaac atcattggca a
//
