LOCUS       sequence-398484-        7864 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 7864)
  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 7864)
  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_37
FEATURES             Location/Qualifiers
     source          1..7864
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(144..160)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(144..160)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(157..179)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(332..920)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(412..431)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1056..1871)
                     /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(1056..1871)
                     /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"
     primer_bind     1779..1798
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1863..1882
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     1992..2008
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1992..2008
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     terminator      2009..2055
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2169..2191
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2183..2200
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2184..2200
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(2614..3465)
                     /codon_start=1
                     /gene="repC"
                     /product="replication protein C of the broad-host-range 
                     plasmid RSF1010 (Scholz et al., 1989)"
                     /label=RSF1010 RepC
                     /translation="MVKPKNKHSLSHVRHDPAHCLAPGLFRALKRGERKRSKLDVTYDY
                     GDGKRIEFSGPEPLGADDLRILQGLVAMAGPNGLVLGPEPKTEGGRQLRLFLEPKWEAV
                     TADAMVVKGSYRALAKEIGAEVDSGGALKHIQDCIERLWKVSIIAQNGRKRQGFRLLSE
                     YASDEADGRLYVALNPLIAQAVMGGGQHVRISMDEVRALDSETARLLHQRLCGWIDPGK
                     TGKASIDTLCGYVWPSEASGSTMRKRRQRVREALPELVALGWTVTEFAAGKYDITRPKA
                     AG"
     CDS             complement(3452..4291)
                     /codon_start=1
                     /gene="repA"
                     /product="replication protein A of the broad-host-range 
                     plasmid RSF1010 (Scholz et al., 1989)"
                     /label=RSF1010 RepA
                     /translation="MATHKPINILEAFAAAPPPLDYVLPNMVAGTVGALVSPGGAGKSM
                     LALQLAAQIAGGPDLLEVGELPTGPVIYLPAEDPPTAIHHRLHALGAHLSAEERQAVAD
                     GLLIQPLIGSLPNIMAPEWFDGLKRAAEGRRLMVLDTLRRFHIEEENASGPMAQVIGRM
                     EAIAADTGCSIVFLHHASKGAAMMGAGDQQQASRGSSVLVDNIRWQSYLSSMTSAEAEE
                     WGVDDDQRRFFVRFGVSKANYGAPFADRWFRRHDGGVLKPAVLERQRKSKGVPRGEA"
     CDS             complement(4802..5773)
                     /codon_start=1
                     /gene="repB"
                     /product="replication protein B of the broad-host-range 
                     plasmid RSF1010 (Scholz et al., 1989)"
                     /label=RSF1010 RepB
                     /translation="MKNDRTLQAIGRQLKAMGCERFDIGVRDATTGQMMNREWSAAEVL
                     QNTPWLKRMNAQGNDVYIRPAEQERHGLVLVDDLSEFDLDDMKAEGREPALVVETSPKN
                     YQAWVKVADAAGGELRGQIARTLASEYDADPASADSRHYGRLAGFTNRKDKHTTRAGYQ
                     PWVLLRESKGKTATAGPALVQQAGQQIEQAQRQQEKARRLASLELPERQLSRHRRTALD
                     EYRSEMAGLVKRFGDDLSKCDFIAAQKLASRGRSAEEIGKAMAEASPALAERKPGHEAD
                     YIERTVSKVMGLPSVQLARAELARAPAPRQRGMDRGGPDFSM"
     oriT            7012..7099
                     /label=RSF1010 oriT
                     /note="origin of transfer of the broad-host-range plasmid 
                     RSF1010 (Scholz et al., 1989)"
     rep_origin      7441..7835
                     /label=RSF1010 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     RSF1010; requires the RSF1010 RepA/B/C proteins for 
                     replication (Scholz et al., 1989)"
ORIGIN
        1 agcatgaaga gcaccccagg atacatagat taccacaact ccgagccctt ccacctagcg
       61 tgctaaccac gcacgctatt gttgtatagg ctaggtggag gctcagtgat gataagtctg
      121 cgatggtgga tgcatgtgtc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
      181 agagggcaca atcctattcc gcgctatccg acaatctcca agacattagg tggagttcag
      241 ttcggcgtat ggcatatgtc gctggaaaga acatgtgagc aaaaggccag caaaaggcca
      301 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
      361 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
      421 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
      481 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
      541 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
      601 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
      661 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
      721 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
      781 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
      841 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
      901 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctctat
      961 tcaacaaagc cgccgtcccg tcaagtcagc gtaaatgggt agggggcttc aaatcgtccg
     1021 ctctgccagt gttacaacca attaacaaat tctgattaga aaaactcatc gagcatcaaa
     1081 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
     1141 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
     1201 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
     1261 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagc
     1321 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
     1381 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga
     1441 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
     1501 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
     1561 ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
     1621 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
     1681 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
     1741 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
     1801 tataaatcag catccatgtt ggaatttaat cgcggcctcg agcaagacgt ttcccgttga
     1861 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat
     1921 gatgatatat ttttatcttg tgcaatgtaa catcagagat tttgagacac aacgtggctt
     1981 tcccccgccg ctctagaact agtggatcca aataaaacga aaggctcagt cgaaagactg
     2041 ggcctttcgt tttatctgtt gtttgtcgca ttatacgaga cgtccaggtt gggatacctg
     2101 aaacaaaacc catcgtacgg ccaaggaagt ctccaataac tgtgatccac cacaagcgcc
     2161 agggttttcc cagtcacgac gttgtaaaac gacggccagt catgcataat ccgcacgcat
     2221 ctggaataag gaagtgccat tccgcctgac cttagcgtgc gaacagcacg ctattgttgt
     2281 atcattactc gcatccattc tcaggctgtc tcgtctcgtc tccgagctct tcaatgagtc
     2341 agggtcacgc gtaggacgcg tcgagtaggg tgacttgtgg ccctttcttt tgttccgcag
     2401 ccacacgccg tgtgaagctg gggagataca tctggaagca aagccaggaa agcggcctat
     2461 ggagctgtgc ggcagcgctc agtaggcaat ttttcaaaat attgttaagc cttttctgag
     2521 catggtattt ttcatggtat taccaattag caggaaaata agccattgaa tataaaagat
     2581 aaaaatgtct tgtttacaat agagtggggg gggtcagcct gccgccttgg gccgggtgat
     2641 gtcgtacttg cccgccgcga actcggttac cgtccagccc agcgcgacca gctccggcaa
     2701 cgcctcgcgc acccgctggc ggcgcttgcg catggtcgaa ccactggcct ctgacggcca
     2761 gacatagccg cacaaggtat ctatggaagc cttgccggtt ttgccggggt cgatccagcc
     2821 acacagccgc tggtgcagca ggcgggcggt ttcgctgtcc agcgcccgca cctcgtccat
     2881 gctgatgcgc acatgctggc cgccacccat gacggcctgc gcgatcaagg ggttcagggc
     2941 cacgtacagg cgcccgtccg cctcgtcgct ggcgtactcc gacagcagcc gaaacccctg
     3001 ccgcttgcgg ccattctggg cgatgatgga taccttccaa aggcgctcga tgcagtcctg
     3061 tatgtgcttg agcgccccac cactatcgac ctctgccccg atttcctttg ccagcgcccg
     3121 atagctacct ttgaccacca tggcatcagc ggtgacggcc tcccacttgg gttccaggaa
     3181 cagccggagc tgccgtccgc cttcggtctt gggttccggg ccaagcacta ggccattagg
     3241 cccagccatg gccaccagcc cttgcaggat gcgcagatca tcagcgccca gcggctccgg
     3301 gccgctgaac tcgatccgct tgccgtcgcc gtagtcatac gtcacgtcca gcttgctgcg
     3361 cttgcgctcg ccccgcttga gggcacggaa caggccgggg gccagacagt gcgccgggtc
     3421 gtgccggacg tggctgaggc tgtgcttgtt cttaggcttc accacggggc acccccttgc
     3481 tcttgcgctg cctctccagc acggcgggct tgagcacccc gccgtcatgc cgcctgaacc
     3541 accgatcagc gaacggtgcg ccatagttgg ccttgctcac accgaagcgg acgaagaacc
     3601 ggcgctggtc gtcgtccaca ccccattcct cggcctcggc gctggtcatg ctcgacaggt
     3661 aggactgcca gcggatgtta tcgaccagta ccgagctgcc ccggctggcc tgctgctggt
     3721 cgcctgcgcc catcatggcc gcgcccttgc tggcatggtg caggaacacg atagagcacc
     3781 cggtatcggc ggcgatggcc tccatgcgac cgatgacctg ggccatgggg ccgctggcgt
     3841 tttcttcctc gatgtggaac cggcgcagcg tgtccagcac catcaggcgg cggccctcgg
     3901 cggcgcgctt gaggccgtcg aaccactccg gggccatgat gttgggcagg ctgccgatca
     3961 gcggctggat cagcaggccg tcagccacgg cttgccgttc ctcggcgctg aggtgcgccc
     4021 caagggcgtg caggcggtga tgaatggcgg tgggcgggtc ttcggcgggc aggtagatca
     4081 ccgggccggt gggcagttcg cccacctcca gcagatccgg cccgcctgca atctgtgcgg
     4141 ccagttgcag ggccagcatg gatttaccgg caccaccggg cgacaccagc gccccgaccg
     4201 taccggccac catgttgggc aaaacgtagt ccagcggtgg cggcgctgct gcgaacgcct
     4261 ccagaatatt gataggctta tgggtagcca ttgattgcct cctttgcagg cagttggtgg
     4321 ttaggcgctg gcggggtcac tacccccgcc ctgcgccgct ctgagttctt ccaggcactc
     4381 gcgcagcgcc tcgtattcgt cgtcggtcag ccagaacttg cgctgacgca tccctttggc
     4441 cttcatgcgc tcggcatatc gcgcttggcg tacagcgtca gggctggcca gcaggtcgcc
     4501 ggtctgcttg tccttttggt ctttcatatc agtcaccgag aaacttgccg gggccgaaag
     4561 gcttgtcttc gcggaacaag gacaaggtgc agccgtcaag gttaaggctg gccatatcag
     4621 cgactgaaaa gcggccagcc tcggccttgt ttgacgtata accaaagcca ccgggcaacc
     4681 aatagccctt gtcacttttg atcaggtaga ccgaccctga agcgcttttt tcgtattcca
     4741 taaaaccccc ttctgtgcgt gagtactcat agtataacag gcgtgagtac caacgcaagc
     4801 actacatgct gaaatctggc ccgcccctgt ccatgcctcg ctggcggggt gccggtgccc
     4861 gtgccagctc ggcccgcgca agctggacgc tgggcagacc catgaccttg ctgacggtgc
     4921 gctcgatgta atccgcttcg tggccgggct tgcgctctgc cagcgctggg ctggcctcgg
     4981 ccatggcctt gccgatttcc tcggcactgc ggccccggct ggccagcttc tgcgcggcga
     5041 taaagtcgca cttgctgagg tcatcaccga agcgcttgac cagcccggcc atctcgctgc
     5101 ggtactcgtc cagcgccgtg cgccggtggc ggctaagctg ccgctcgggc agttcgaggc
     5161 tggccagcct gcgggccttc tcctgctgcc gctgggcctg ctcgatctgc tggccagcct
     5221 gctgcaccag cgccgggcca gcggtggcgg tcttgccctt ggattcacgc agcagcaccc
     5281 acggctgata accggcgcgg gtggtgtgct tgtccttgcg gttggtgaag cccgccaagc
     5341 ggccatagtg gcggctgtcg gcgctggccg ggtcggcgtc gtactcgctg gccagcgtcc
     5401 gggcaatctg cccccgaagt tcaccgcctg cggcgtcggc caccttgacc catgcctgat
     5461 agttcttcgg gctggtttcc actaccaggg caggctcccg gccctcggct ttcatgtcat
     5521 ccaggtcaaa ctcgctgagg tcgtccacca gcaccagacc atgccgctcc tgctcggcgg
     5581 gcctgatata cacgtcattg ccctgggcat tcatccgctt gagccatggc gtgttctgga
     5641 gcacttcggc ggctgaccat tcccggttca tcatctggcc ggtggtggcg tccctgacgc
     5701 cgatatcgaa gcgctcacag cccatggcct tgagctgtcg gcctatggcc tgcaaagtcc
     5761 tgtcgttctt catcgggcca ccaagcgcag ccagatcgag ccgtcctcgg ttgtcagtgg
     5821 cgtcaggtcg agcaagagca acgatgcgat cagcagcacc accgtaggca tcatggaagc
     5881 cagcatcacg gttagccata gcttccagtg ccacccccgc gacgcgctcc gggcgctctg
     5941 cgcggcgctg ctcacctcgg cggctacctc ccgcaactct ttggccagct ccacccatgc
     6001 cgcccctgtc tggcgctggg ctttcagcca ctccgccgcc tgcgcctcgc tggcctgctg
     6061 ggtctggctc atgacctgcc gggcttcgtc ggccagtgtc gccatgctct gggccagcgg
     6121 ttcgatctgc tccgctaact cgttgatgcc tctggatttc ttcactctgt cgattgcgtt
     6181 catggtctat tgcctcccgg tattcctgta agtcgatgat ctgggcgttg gcggtgtcga
     6241 tgttcagggc cacgtctgcc cggtcggtgc ggatgccccg gccttccatc tccaccacgt
     6301 tcggccccag gtgaacaccg ggcaggcgct cgatgccctg cgcctcaagt gttctgtggt
     6361 caatgcgggc gtcgtggcca gcccgctcta atgcccggtt ggcatggtcg gcccatgcct
     6421 cgcgggtctg ctcaagccat gccttgggct tgagcgcttc ggtcttctgt gccccgccct
     6481 tctccggggt cttgccgttg taccgcttga accactgagc ggcgggccgc tcgatgccgt
     6541 cattgatccg ctcggagatc atcaggtggc agtgcgggtt ctcgccgcca ccggcatgga
     6601 tggccagcgt atacggcagg cgctcggcac cggtcaggtg ctgggcgaac tcggacgcca
     6661 gcgccttctg ctggtcgagg gtcagctcga ccggcagggc aaattcgacc tccttgaaca
     6721 gccgcccatt ggcgcgttca tacaggtcgg cagcatccca gtagtcggcg ggccgctcga
     6781 cgaactccgg catgtgcccg gattcggcgt gcaagacttc atccatgtcg cgggcatact
     6841 tgccttcgcg ctggatgtag tcggccttgg ccctggccga ttggccgccc gacctgctgc
     6901 cggttttcgc cgtaaggtga taaatcgcca tgctgcctcg ctgttgcttt tgcttttcgg
     6961 ctccatgcaa tggccctcgg agagcgcacc gcccgaaggg tggccgttag gccagtttct
     7021 cgaagagaaa ccggtaagtg cgccctcccc tacaaagtag ggtcgggatt gccgccgctg
     7081 tgcctccatg atagcctacg agacagcaca ttaacaatgg ggtgtcaaga tggttaaggg
     7141 gagcaacaag gcggcggatc ggctggccaa gctcgaagaa caacgagcgc gaatcaatgc
     7201 cgaaattcag cgggtgcggg caagggaaca gcagcaagag cgcaagaacg aaacaaggcg
     7261 caaggtgctg gtgggggcca tgattttggc caaggtgaac agcagcgagt ggccggagga
     7321 tcggctcatg gcggcaatgg atgcgtacct tgaacgcgac cacgaccgcg ccttgttcgg
     7381 tctgccgcca cgccagaagg atgagccggg ctgaatgatc gaccgagaca ggccctgcgg
     7441 ggctgcacac gcgcccccac ccttcgggta gggggaaagg ccgctaaagc ggctaaaagc
     7501 gctccagcgt atttctgcgg ggtttggtgt ggggtttagc gggctttgcc cgcctttccc
     7561 cctgccgcgc agcggtgggg cggtgtgtag cctagcgcag cgaatagacc agctatccgg
     7621 cctctggccg ggcatattgg gcaagggcag cagcgcccca caagggcgct gataaccgcg
     7681 cctagtggat tattcttaga taatcatgga tggatttttc caacaccccg ccagcccccg
     7741 cccctgctgg gtttgcaggt ttgggggcgt gacagttatt gcaggggttc gtgacagtta
     7801 ttgcaggggg gcgtgacagt tattgcaggg gttcgtgaca gttagtacgg gagtgacggg
     7861 cgct
//
