LOCUS       sequence-398544-        6745 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 plasmid, barcoded ORI with gentamicin resistance cassette 
            and mScarlet-I reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 936 to 970)
  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 6745)
  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: pGL2_172
FEATURES             Location/Qualifiers
     source          1..6745
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(9..14,15..31,32..37)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             227..760
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        936..970
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     oriT            1896..2004
                     /note="incP origin of transfer"
     primer_bind     complement(2158..2174)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2158..2174)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2171..2193)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      2364..2752
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(2901..2995)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(3016..3675)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3590..3609
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3676..3778)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     3865..3887
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3879..3896
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3880..3896
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             4637..6079
                     /codon_start=1
                     /gene="rep"
                     /product="replication initiator protein from the 
                     Streptomyces ghanaensis plasmid pSG5"
                     /label=pSG5 Rep
                     /translation="MWKPGEATWGNTCRCNNVHTCPWCMSRILAVRGSNVQLAADGLAD
                     AGYGLHLGTNTLRHFERMAFGTVRKGMRHGLVAVLHDGWKGAYGSSGRRWRTMRDDFGI
                     IGYERAFEDTFGWGSGWHLHWHTLWVTREVLGPDAQAAFRDALAGAWAAGVESAGGYTV
                     SETCDRPGCSCEGKGHGTDVRPLNGADAADGDAGKQARYLYKDGDKTKGGVAKIGLELA
                     GQNFKAGRGDDRMGPLDLGDAAAAELQRLRRPGPFVEKYREREFGVFQVRKHYRSQNLN
                     RLIKELGIQQDVRTEEEITDDTEGLVAIAVIPAYIWYRYIARVAGRRLDLIKVAETYGL
                     PGVRRLVESWGLVWGKDVLDPPAPEAPAAPGDLDADQMRFEVMSEEEAAFREARRKANE
                     ARTEELAASLDRVRQPKKEAIRPTISLRKRLKPKPVTVDVKTPPPGAASPVCRRCKGKL
                     APVLQPWGQHPGDCLRVDTAVA"
ORIGIN
        1 gcaaggagtt gacataagcc tgttcggttc gtaaactgta atgcaagtag cgtatgcgct
       61 cacgcaactg gtccagaacc ttgaccgaac gcagcggtgg taacggcgca gtggcggttt
      121 tcatggcttg ttatgactgt ttttttgtac agtctatgcc tcgggcatcc aagcagcaag
      181 cgcgttacgc cgtgggtcga tgtttgatgt tatggagcag caacgaatgt tacgcagcag
      241 caacgatgtt acgcagcagg gcagtcgccc taaaacaaag ttaggtggct caagtatggg
      301 catcattcgc acatgtaggc tcggccctga ccaagtcaaa tccatgcggg ctgctcttga
      361 tcttttcggt cgtgagttcg gagacgtagc cacctactcc caacatcagc cggactccga
      421 ttacctcggg aacttgctcc gtagtaagac attcatcgcg cttgctgcct tcgaccaaga
      481 agcggttgtt ggcgctctcg cggcttacgt tctgcccaag tttgagcagc cgcgtagtga
      541 gatctatatc tatgatctcg cagtctccgg agagcaccgg aggcagggca ttgccaccgc
      601 gctcatcaat ctcctcaagc atgaggccaa cgcgcttggt gcttatgtga tctacgtgca
      661 agcagattac ggtgacgatc ccgcagtggc tctctataca aagttgggca tacgggaaga
      721 agtgatgcac tttgatatcg acccaagtac cgccacctaa gcttgagcgc atgctcgagt
      781 acttcgcctg gaccatgctc gccgtcgtct tcggcttcct cctcttcgtc aacctcgcct
      841 acgtccccct cggccactgg gccgaggcct tcgccggctt cttcaaattt tcagggctgc
      901 cgcaccccat cgactggggg ctcgctatac aggagttgac ggctagctca gtcctaggta
      961 cagtgctagc tactaggagg aaaaaaatgg tgagtaaggg tgaagccgtg attaaagagt
     1021 tcatgcgctt caaagtgcat atggagggct caatgaacgg ccatgaattt gaaattgaag
     1081 gtgaaggaga aggccgtccg tatgagggca cccagaccgc caaactgaaa gtcacaaagg
     1141 gtggcccgct gccgttttcg tgggatattt tatcccctca attcatgtac ggcagccggg
     1201 cgtttattaa acacccggcg gatatcccgg attactataa gcaatctttt ccagaaggct
     1261 ttaaatggga gcgcgtgatg aattttgaag acggaggtgc tgttaccgtg acccaggaca
     1321 cgagcttaga agacggtacc ttgatctata aggtaaagct gcgtggtacc aatttcccgc
     1381 cggatgggcc agttatgcag aaaaagacta tgggctggga agcatcgacg gagcgtcttt
     1441 accccgaaga cggcgtcctg aaaggcgata ttaaaatggc cctgcgactg aaagacggcg
     1501 gtcgctacct tgcagatttc aaaacaacgt ataaagcgaa aaaacctgtg cagatgcccg
     1561 gggcgtacaa cgttgatcgc aaactagata tcacttcaca caacgaagat tacacggtag
     1621 tcgaacagta tgaacgtagc gaggggcgcc atagcaccgg aggcatggat gaactctata
     1681 aataagcttc gctcgtgctc gccgcgacct tcctcttcat gcacctcttc ggcatcgggc
     1741 tgcacaacgc tacagaggag cttttccgct gcataaccct gcttcggggt cattatagcg
     1801 attttttcgg tatatccatc ctttttcgca cgatatacag gattttgcca aagggttcgt
     1861 gtagactttc cttggtgtat ccaacggcgt cagccgggca ggataggtga agtaggccca
     1921 cccgcgagcg ggtgttcctt cttcactgtc ccttattcgc acctggcggt gctcaacggg
     1981 aatcctgctc tgcgaggctg gccgtacgct aggtatactt gagaccaccc caggatacat
     2041 agattaccac aactccgagc ccttccacct agcgtgctaa ccacgcacgc tattgttgta
     2101 taggctaggt ggaggctcag tgatgataag tctgcgatgg tggatgcatg tgtcatggtc
     2161 atagctgttt cctgtgtgaa attgttatcc gctcagaggg cacaatccta ttccgcgcta
     2221 tccgacaatc tccaagacat taggtggagt tcagttcggc gtatggcata tgtcgctgga
     2281 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     2341 gcgttacagc tgggcgcgcc ccatgtcagc cgttaagtgt tcctgtgtca ctcaaaattg
     2401 ctttgagagg ctctaagggc ttctcagtgc gttacatccc tggcttgttg tccacaaccg
     2461 ttaaacctta aaggctttaa aagccttata tattcttttt tttcttataa aacttaaaac
     2521 cttagaggct atttaagttg ctgatttata ttaattttat tgttcaaaca tgagagctta
     2581 gtacgtgaaa catgagagct tagtacgtta gccatgagag cttagtacgt tagccatgag
     2641 ggtttagttc gttaaacatg agagcttagt acgttaaaca tgagagctta gtacgtgaaa
     2701 catgagagct tagtacgtac tatcaacagg ttgaactgct gatcttcaga tcggcgcgcc
     2761 ggccggccga agatcctttg atcttttcta cggggtctga cgctctattc aacaaagccg
     2821 ccgtcccgtc aagtcagcgt aaatgggtag ggggcttcaa atcgtccccc catacgatat
     2881 aagttgttac tagtgcttgg attctcacca ataaaaaacg cccggcggca accgagcgtt
     2941 ctgaacaaat ccagatggag ttctgaggtc attactggat ctatcaacag gagtccaagc
     3001 gagctcgata tcaaattacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt
     3061 aagcattctg ccgacatgga agccatcaca aacggcatga tgaacctgaa tcgccagcgg
     3121 catcagcacc ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa
     3181 gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga
     3241 gacgaaaaac atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca
     3301 cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca
     3361 gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc
     3421 ccatatcacc agctcaccgt ctttcattgc catacgaaat tccggatgag cattcatcag
     3481 gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtctt
     3541 taaaaaggcc gtaatatcca gctgaacggt ctggttatag gtacattgag caactgactg
     3601 aaatgcctca aaatgttctt tacgatgcca ttgggatata tcaacggtgg tatatccagt
     3661 gatttttttc tccattttag cttccttagc tcctgaaaat ctcgataact caaaaaatac
     3721 gcccggtagt gatcttattt cattatggtg aaagttggaa cctcttacgt gccgatcaac
     3781 gtctcattga tacctgaaac aaaacccatc gtacggccaa ggaagtctcc aataactgtg
     3841 atccaccaca agcgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtcatg
     3901 cataatccgc acgcatctgg aataaggaag tgccattccg cctgacctta gcgtgcgaac
     3961 agcacgctat tgttgtatca ttactcgcat ccattctcag gctgtctcgt ctcgtctccg
     4021 aggtctcagg agaatgagtc agggtcacgc gtaggacgcg tcgagtaggg ttccgtttgt
     4081 tcaaaggtca cgttccgcag ccacacgccg tgtgaagctg gggaggatca acaggaccca
     4141 gaaatggcac gagcccggga ttggcctccc gggctcgccg cccccgaacc ggcgcagcat
     4201 gccgagtgag gacgtttgct ctgtgacgag cgtacgagag aacagcgcgt cgacaccacc
     4261 gcgccccgcc ggaccgaagt gccactgcgg caccccgatc gtccacatcc ccgggaagcg
     4321 ccccaaaatc tactgctcgg acgcctgccg aaagcgcgca aagcgtctca ttgcggacca
     4381 gtcacgcagc gtagcaagca cgactacagc gggtaatccg gacgcgggct ttagtagtaa
     4441 gggaaaatct ccgtccactg caggtcagag cgctgtaact ggaacgggtt ctaccccaaa
     4501 cgagggtgcc gcgacagccg ggcgggagga cccgcgcgac gagcggttcg cccggcggga
     4561 ccggcaccag acggtgtcgc tgaacgaggc gttcaagggc tgcgggaccc ggctcactgc
     4621 cgggacggcc gagctgatgt ggaagccggg cgaggcgacg tggggcaaca cgtgccgctg
     4681 caacaacgtc cacacctgcc cgtggtgcat gagccgcatc ctggccgtac ggggctcgaa
     4741 cgtgcagctc gccgccgacg gcctcgcgga cgccgggtac gggctgcacc ttggcacgaa
     4801 cacgttgcgc cacttcgagc gcatggcgtt cggcaccgtc cgcaagggaa tgcggcacgg
     4861 cctggtcgcg gtcttgcacg acggctggaa gggcgcgtac ggctcgtccg gccgccgctg
     4921 gcgcacgatg cgcgacgact tcgggatcat cggctacgag cgggccttcg aggacacctt
     4981 cggctggggc tccggctggc acctgcactg gcacacgctc tgggtcaccc gcgaggtgct
     5041 cggcccggac gcccaggccg cgttccgcga cgcactcgcc ggagcctggg ccgccggggt
     5101 cgaatccgcg ggcggctaca ccgtcagcga gacgtgcgac cggcccggtt gctcctgtga
     5161 gggcaagggc cacggcaccg acgtgcgccc actcaacggc gcggacgccg ctgacggcga
     5221 tgcgggcaag caggcccgct acctctacaa ggacggcgac aagaccaagg gcggcgtcgc
     5281 caagatcggc ctggagctgg ccggacagaa cttcaaggcc ggtcgcggcg acgaccgcat
     5341 gggaccgctc gacctgggcg acgcggcggc cgccgagctg cagcggcttc gtcggcccgg
     5401 cccgttcgtc gagaagtacc gcgagcgcga gttcggcgtc ttccaggtcc gcaagcacta
     5461 ccggtcgcag aacctgaacc ggctgatcaa ggaactcggc atccagcagg acgtccgcac
     5521 cgaggaggag atcaccgacg acaccgaggg cctggtcgcc atcgcggtca tcccggccta
     5581 catctggtac cgatacatcg cccgcgtcgc cggacgtcgc ctcgacctca tcaaggtcgc
     5641 cgagacgtac ggcctaccgg gcgtccggcg gctcgtcgag tcctggggcc tggtgtgggg
     5701 caaggacgtc ctggacccgc ccgccccgga agcgccggcc gcgccgggcg accttgacgc
     5761 tgaccagatg aggttcgagg tcatgtccga ggaggaagct gctttccgcg aggcgcgccg
     5821 gaaggccaac gaggcccgca cggaggagct ggccgcttcg ctcgaccgag tgcgtcagcc
     5881 gaagaaggag gcgatccgcc cgacgatcag ccttcggaag cgcctgaagc cgaagccggt
     5941 caccgtcgac gtcaagactc ccccgcccgg cgccgcgtcg cccgtgtgcc gccgctgcaa
     6001 gggcaagctc gccccggtac tccagccctg gggccagcac cccggagact gcctccgcgt
     6061 cgacacagcg gtcgcgtagg gccgctgagg gaccgccatc agaccgccac cccggacatc
     6121 cgggtggcgg tctgatggcg gtcccggcgc gaaagccgag ggtctgcctg ccgtgaggtg
     6181 gccggcggac ctgggttact gctggaatcc cgcgcccagt ccgagcgtcc gccggcgctt
     6241 ttcacctggc tccgggtgct cgtcgatgag caccaggccg tcgacgacga cgcgtccgcc
     6301 tgcctgcttt tggccggcgg gccgggccgc ggttgtccgc cgggtcggac cggtcccttg
     6361 gtcgaccgat gcctcggcca cgacgatgct gggcccgaag cggtacgcga cccgtgccgg
     6421 gccgcagcgg atcgcggcag caggagctcc ccgcccgcca cggctggctg gatggtcgcc
     6481 tcacgcgccc gcaggggtga cggcgtgggt gcggcggtca gccccgtgat cgggcgtcag
     6541 acggccgctg agggaccgcg accacccctc ccttcccgcc tgccgtcttt caccgcccgg
     6601 ccggtccgca gacgccgtac ggccaggcca cggaggatta cgggcagatg gcgcccaccc
     6661 ggcacccggg ctggggcacc ctggacagca cgaaggtccg ccccactcac cggggcgggc
     6721 cttcgcgttg agctagagtt cgcta
//
