LOCUS       sequence-399683-        5958 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 916 to 950)
  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 5958)
  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_174
FEATURES             Location/Qualifiers
     source          1..5958
                     /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             207..740
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        916..950
                     /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            1876..1984
                     /note="incP origin of transfer"
     primer_bind     complement(2138..2154)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2138..2154)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2151..2173)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      2344..2732
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(2881..2975)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(2996..3655)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3570..3589
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3656..3758)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     3845..3867
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3859..3876
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3860..3876
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
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 tacgcagcag caacgatgtt acgcagcagg
      241 gcagtcgccc taaaacaaag ttaggtggct caagtatggg catcattcgc acatgtaggc
      301 tcggccctga ccaagtcaaa tccatgcggg ctgctcttga tcttttcggt cgtgagttcg
      361 gagacgtagc cacctactcc caacatcagc cggactccga ttacctcggg aacttgctcc
      421 gtagtaagac attcatcgcg cttgctgcct tcgaccaaga agcggttgtt ggcgctctcg
      481 cggcttacgt tctgcccaag tttgagcagc cgcgtagtga gatctatatc tatgatctcg
      541 cagtctccgg agagcaccgg aggcagggca ttgccaccgc gctcatcaat ctcctcaagc
      601 atgaggccaa cgcgcttggt gcttatgtga tctacgtgca agcagattac ggtgacgatc
      661 ccgcagtggc tctctataca aagttgggca tacgggaaga agtgatgcac tttgatatcg
      721 acccaagtac cgccacctaa gcttgagcgc atgctcgagt acttcgcctg gaccatgctc
      781 gccgtcgtct tcggcttcct cctcttcgtc aacctcgcct acgtccccct cggccactgg
      841 gccgaggcct tcgccggctt cttcaaattt tcagggctgc cgcaccccat cgactggggg
      901 ctcgctatac aggagttgac ggctagctca gtcctaggta cagtgctagc tactaggagg
      961 aaaaaaatgg tgagtaaggg tgaagccgtg attaaagagt tcatgcgctt caaagtgcat
     1021 atggagggct caatgaacgg ccatgaattt gaaattgaag gtgaaggaga aggccgtccg
     1081 tatgagggca cccagaccgc caaactgaaa gtcacaaagg gtggcccgct gccgttttcg
     1141 tgggatattt tatcccctca attcatgtac ggcagccggg cgtttattaa acacccggcg
     1201 gatatcccgg attactataa gcaatctttt ccagaaggct ttaaatggga gcgcgtgatg
     1261 aattttgaag acggaggtgc tgttaccgtg acccaggaca cgagcttaga agacggtacc
     1321 ttgatctata aggtaaagct gcgtggtacc aatttcccgc cggatgggcc agttatgcag
     1381 aaaaagacta tgggctggga agcatcgacg gagcgtcttt accccgaaga cggcgtcctg
     1441 aaaggcgata ttaaaatggc cctgcgactg aaagacggcg gtcgctacct tgcagatttc
     1501 aaaacaacgt ataaagcgaa aaaacctgtg cagatgcccg gggcgtacaa cgttgatcgc
     1561 aaactagata tcacttcaca caacgaagat tacacggtag tcgaacagta tgaacgtagc
     1621 gaggggcgcc atagcaccgg aggcatggat gaactctata aataagcttc gctcgtgctc
     1681 gccgcgacct tcctcttcat gcacctcttc ggcatcgggc tgcacaacgc tacagaggag
     1741 cttttccgct gcataaccct gcttcggggt cattatagcg attttttcgg tatatccatc
     1801 ctttttcgca cgatatacag gattttgcca aagggttcgt gtagactttc cttggtgtat
     1861 ccaacggcgt cagccgggca ggataggtga agtaggccca cccgcgagcg ggtgttcctt
     1921 cttcactgtc ccttattcgc acctggcggt gctcaacggg aatcctgctc tgcgaggctg
     1981 gccgtacgct aggtatactt gagaccaccc caggatacat agattaccac aactccgagc
     2041 ccttccacct agcgtgctaa ccacgcacgc tattgttgta taggctaggt ggaggctcag
     2101 tgatgataag tctgcgatgg tggatgcatg tgtcatggtc atagctgttt cctgtgtgaa
     2161 attgttatcc gctcagaggg cacaatccta ttccgcgcta tccgacaatc tccaagacat
     2221 taggtggagt tcagttcggc gtatggcata tgtcgctgga aagaacatgt gagcaaaagg
     2281 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgttacagc tgggcgcgcc
     2341 ccatgtcagc cgttaagtgt tcctgtgtca ctcaaaattg ctttgagagg ctctaagggc
     2401 ttctcagtgc gttacatccc tggcttgttg tccacaaccg ttaaacctta aaggctttaa
     2461 aagccttata tattcttttt tttcttataa aacttaaaac cttagaggct atttaagttg
     2521 ctgatttata ttaattttat tgttcaaaca tgagagctta gtacgtgaaa catgagagct
     2581 tagtacgtta gccatgagag cttagtacgt tagccatgag ggtttagttc gttaaacatg
     2641 agagcttagt acgttaaaca tgagagctta gtacgtgaaa catgagagct tagtacgtac
     2701 tatcaacagg ttgaactgct gatcttcaga tcggcgcgcc ggccggccga agatcctttg
     2761 atcttttcta cggggtctga cgctctattc aacaaagccg ccgtcccgtc aagtcagcgt
     2821 aaatgggtag ggggcttcaa atcgtccccc catacgatat aagttgttac tagtgcttgg
     2881 attctcacca ataaaaaacg cccggcggca accgagcgtt ctgaacaaat ccagatggag
     2941 ttctgaggtc attactggat ctatcaacag gagtccaagc gagctcgata tcaaattacg
     3001 ccccgccctg ccactcatcg cagtactgtt gtaattcatt aagcattctg ccgacatgga
     3061 agccatcaca aacggcatga tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt
     3121 gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt
     3181 ttaaatcaaa actggtgaaa ctcacccagg gattggctga gacgaaaaac atattctcaa
     3241 taaacccttt agggaaatag gccaggtttt caccgtaaca cgccacatct tgcgaatata
     3301 tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa aacgtttcag
     3361 tttgctcatg gaaaacggtg taacaagggt gaacactatc ccatatcacc agctcaccgt
     3421 ctttcattgc catacgaaat tccggatgag cattcatcag gcgggcaaga atgtgaataa
     3481 aggccggata aaacttgtgc ttatttttct ttacggtctt taaaaaggcc gtaatatcca
     3541 gctgaacggt ctggttatag gtacattgag caactgactg aaatgcctca aaatgttctt
     3601 tacgatgcca ttgggatata tcaacggtgg tatatccagt gatttttttc tccattttag
     3661 cttccttagc tcctgaaaat ctcgataact caaaaaatac gcccggtagt gatcttattt
     3721 cattatggtg aaagttggaa cctcttacgt gccgatcaac gtctcattga tacctgaaac
     3781 aaaacccatc gtacggccaa ggaagtctcc aataactgtg atccaccaca agcgccaggg
     3841 ttttcccagt cacgacgttg taaaacgacg gccagtcatg cataatccgc acgcatctgg
     3901 aataaggaag tgccattccg cctgacctta gcgtgcgaac agcacgctat tgttgtatca
     3961 ttactcgcat ccattctcag gctgtctcgt ctcgtctccg aggtctcagg agaatgagtc
     4021 agggtcacgc gtaggacgcg tcgagtaggg tccgagagtt aaggccacta tgttccgcag
     4081 ccacacgccg tgtgaagctg gggagatggt aaatctgcgc agacagccct gtgcagctga
     4141 aacgcggtta cgtatagctt gccatatgtc tagccatacg taaccgcagg taaaaggcat
     4201 atttttcgcg tgtcatggct agtaaataac accggtgtca tttagagtca gggaaagaca
     4261 atgaaaaacg aagaaagcca ccgggcggca acccgatgac tttcgcttat cacccagcac
     4321 acacctggga gaaatcacgg tcatgagttt acagactcat gcgcagaatg cgcacactaa
     4381 aacacctacc cgcgtcgagc gcgaccgtgg tggactggac aacaccccag catctgccag
     4441 tgaccgcgac cttttacgcg atcatctagg ccgcgatgta ctccacggtt cagtcacacg
     4501 agactttaaa aaggcctatc gacgcaacgc tgacggcacg aactcgccgc gtatgtatcg
     4561 cttcgagact gatgctttag gacggtgcga gtacgccatg ctcaccacca agcagtacgc
     4621 cgccgtcctg gtcgtagacg ttgaccaagt aggtaccgca ggcggtgacc ccgcagactt
     4681 aaacccgtac gtccgcgacg tggtgcgctc actgattact catagcgtcg ggccagcctg
     4741 ggtgggtatt aacccaacta acggcaaagc ccagttcata tggcttattg accctgtcta
     4801 cgctgaccgt aacggtaaat ctgcgcagat gaagcttctt gcagcaacca cgcgtgtgct
     4861 gggtgagctt ttagaccatg acccgcactt ttcccaccgc tttagccgca acccgttcta
     4921 cacaggcaaa gcccctaccg cttatcgttg gtataggcag cacaaccggg tgatgcgcct
     4981 tggagacttg ataaagcagg taagggatat ggcaggacac gaccagttca accccacccc
     5041 acgccagcaa ttcagctctg gccgcgaact tatcaacgcg gtcaagaccc gccgtgaaga
     5101 agcccaagca ttcaaagcac tcgcccagga cgtagacgcg gaaatcgccg gtggcctcga
     5161 ccagtatgac ccggaactta tcgacggtgt gcgtgtgctc tggattgtcc aaggaaccgc
     5221 agcacgcgac gaaacagcct ttagacatgc gcttaagact ggccaccgct tgcgccagca
     5281 aggccaacgc ctgacagacg cagcaatcat cgacgcctat gagcacgcct acaacgtcgc
     5341 acacacccac ggcggtgcag gccgcgacaa cgagatgcca cccatgcgcg accgccaaac
     5401 catggcaagg cgcgtgcgcg ggtatgtcgc ccaatccaag agcgaaacct acagcggctc
     5461 taacgcacca ggtaaagcca ccagcagcga gcggaaagcc ttggccacga tgggacgcag
     5521 aggcggacaa aaagccgcac aacgctggaa aacagacccc gagggcaaat atgcgcaagc
     5581 acaaaggtcg aagcttgaaa agacgcaccg taagaaaaag gctcaaggac gatctacgaa
     5641 gtcccgtatt agccaaatgg tgaacgatca gtatttccag acagggacag ttcccacgtg
     5701 ggctgaaata ggggcagagg taggagtctc tcgcgccacg gttgctaggc atgtcgcgga
     5761 gctaaagaaa agcggtgact atccggacgt ttaaggcgtc tcataccgta agcaatatac
     5821 ggttcccctg ccgttaggca gttagataaa acctcacttg aagaaaacct tgaggggcag
     5881 ggcagcttat atgcttcaaa gcatgacttc ctctgttctc ctagacctcg caaccctccg
     5941 ccataacctc acccgcta
//
