LOCUS       sequence-397963-        4176 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 4176)
  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 4176)
  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_9
FEATURES             Location/Qualifiers
     source          1..4176
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     557..576
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     641..660
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     770..786
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     770..786
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     terminator      787..833
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     947..969
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     961..978
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     962..978
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      1684..1906
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             1954..2904
                     /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"
     primer_bind     complement(3098..3114)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3098..3114)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3111..3133)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(3286..3874)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3366..3385)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4010..4176,1..649))
                     /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(4010..4176,1..649))
                     /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 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac
       61 catgagtgac gactgaatcc ggtgagaatg gcaaaagctt atgcatttct ttccagactt
      121 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat
      181 tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac
      241 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac
      301 ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga
      361 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt
      421 ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc
      481 catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac
      541 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg
      601 aatttaatcg cggcctcgag caagacgttt cccgttgaat atggctcata acaccccttg
      661 tattactgtt tatgtaagca gacagtttta ttgttcatga tgatatattt ttatcttgtg
      721 caatgtaaca tcagagattt tgagacacaa cgtggctttc ccccgccgct ctagaactag
      781 tggatccaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt
      841 ttgtcgcatt atacgagacg tccaggttgg gatacctgaa acaaaaccca tcgtacggcc
      901 aaggaagtct ccaataactg tgatccacca caagcgccag ggttttccca gtcacgacgt
      961 tgtaaaacga cggccagtca tgcataatcc gcacgcatct ggaataagga agtgccattc
     1021 cgcctgacct tagcgtgcga acagcacgct attgttgtat cattactcgc atccattctc
     1081 aggctgtctc gtctcgtctc cgagctcttc aatgagtcag ggtcacgcgt aggacgcgtc
     1141 gagtagggtc ccgatagctg agaaagagtg ttccgcagcc acacgccgtg tgaagctggg
     1201 gaggaccccg ttgatgatac cgctgcctta ctgggtgcat tagccagtct gaatgacctg
     1261 tcacgggata atccgaagtg gtcagactgg aaaatcagag ggcaggaact gctgaacagc
     1321 aaaaagtcag atagcaccac atagcagacc cgccataaaa cgccctgaga agcccgtgac
     1381 gggcttttct tgtattatgg gtagtttcct tgcatgaatc cataaaaggc gcctgtagtg
     1441 ccatttaccc ccattcactg ccagagccgt gagcgcagcg aactgaatgt cacgaaaaag
     1501 acagcgactc aggtgcctga tggtcggaga caaaaggaat attcagcgat ttgcccgatt
     1561 gcggccgcaa ccgagcttgc gagggtgcta cttaagcctt tagggtttta aggtctgttt
     1621 tgtagaggag caaacagcgt ttgcgacatc cttttgtaat actgcggaac tgactaaagt
     1681 agtgagttat acacagggct gggatctatt ctttttatct ttttttattc tttctttatt
     1741 ctataaatta taaccacttg aatataaaca aaaaaaacac acaaaggtct agcggaattt
     1801 acagagggtc tagcagaatt tacaagtttt ccagcaaagg tctagcagaa tttacagata
     1861 cccacaactc aaaggaaaag gactagtaat tatcattgac tagcccatct caattggtat
     1921 agtgattaaa atcacctaga ccaattgaga tgtatgtctg aattagttgt tttcaaagca
     1981 aatgaactag cgattagtcg ctatgactta acggagcatg aaaccaagct aattttatgc
     2041 tgtgtggcac tactcaaccc cacgattgaa aaccctacaa ggaaagaacg gacggtatcg
     2101 ttcacttata accaatacgt tcagatgatg aacatcagta gggaaaatgc ttatggtgta
     2161 ttagctaaag caaccagaga gctgatgacg agaactgtgg aaatcaggaa tcctttggtt
     2221 aaaggctttg agattttcca gtggacaaac tatgccaagt tctcaagcga aaaattagaa
     2281 ttagttttta gtgaagagat attgccttat cttttccagt taaaaaaatt cataaaatat
     2341 aatctggaac atgttaagtc ttttgaaaac aaatactcta tgaggattta tgagtggtta
     2401 ttaaaagaac taacacaaaa gaaaactcac aaggcaaata tagagattag ccttgatgaa
     2461 tttaagttca tgttaatgct tgaaaataac taccatgagt ttaaaaggct taaccaatgg
     2521 gttttgaaac caataagtaa agatttaaac acttacagca atatgaaatt ggtggttgat
     2581 aagcgaggcc gcccgactga tacgttgatt ttccaagttg aactagatag acaaatggat
     2641 ctcgtaaccg aacttgagaa caaccagata aaaatgaatg gtgacaaaat accaacaacc
     2701 attacatcag attcctacct acataacgga ctaagaaaaa cactacacga tgctttaact
     2761 gcaaaaattc agctcaccag ttttgaggca aaatttttga gtgacatgca aagtaagtat
     2821 gatctcaatg gttcgttctc atggctcacg caaaaacaac gaaccacact agagaacata
     2881 ctggctaaat acggaaggat ctgaggttct tatggctctt gtatctatca gtgaagcatc
     2941 aagactaaca cgctagcatg aagagcaccc caggatacat agattaccac aactccgagc
     3001 ccttccacct agcgtgctaa ccacgcacgc tattgttgta taggctaggt ggaggctcag
     3061 tgatgataag tctgcgatgg tggatgcatg tgtcatggtc atagctgttt cctgtgtgaa
     3121 attgttatcc gctcagaggg cacaatccta ttccgcgcta tccgacaatc tccaagacat
     3181 taggtggagt tcagttcggc gtatggcata tgtcgctgga aagaacatgt gagcaaaagg
     3241 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     3301 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     3361 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     3421 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     3481 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     3541 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     3601 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     3661 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     3721 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     3781 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     3841 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     3901 gtctgacgct ctattcaaca aagccgccgt cccgtcaagt cagcgtaaat gggtaggggg
     3961 cttcaaatcg tccgctctgc cagtgttaca accaattaac aaattctgat tagaaaaact
     4021 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     4081 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     4141 gatcctggta tcggtctgcg attccgactc gtccaa
//
