LOCUS       sequence-399666-        5877 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 5877)
  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_165
FEATURES             Location/Qualifiers
     source          1..5877
                     /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"
     rep_origin      4606..4828
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             4876..5826
                     /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"
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 tcccgatagc
     4081 tgagaaagag tgttccgcag ccacacgccg tgtgaagctg gggaggaccc cgttgatgat
     4141 accgctgcct tactgggtgc attagccagt ctgaatgacc tgtcacggga taatccgaag
     4201 tggtcagact ggaaaatcag agggcaggaa ctgctgaaca gcaaaaagtc agatagcacc
     4261 acatagcaga cccgccataa aacgccctga gaagcccgtg acgggctttt cttgtattat
     4321 gggtagtttc cttgcatgaa tccataaaag gcgcctgtag tgccatttac ccccattcac
     4381 tgccagagcc gtgagcgcag cgaactgaat gtcacgaaaa agacagcgac tcaggtgcct
     4441 gatggtcgga gacaaaagga atattcagcg atttgcccga ttgcggccgc aaccgagctt
     4501 gcgagggtgc tacttaagcc tttagggttt taaggtctgt tttgtagagg agcaaacagc
     4561 gtttgcgaca tccttttgta atactgcgga actgactaaa gtagtgagtt atacacaggg
     4621 ctgggatcta ttctttttat ctttttttat tctttcttta ttctataaat tataaccact
     4681 tgaatataaa caaaaaaaac acacaaaggt ctagcggaat ttacagaggg tctagcagaa
     4741 tttacaagtt ttccagcaaa ggtctagcag aatttacaga tacccacaac tcaaaggaaa
     4801 aggactagta attatcattg actagcccat ctcaattggt atagtgatta aaatcaccta
     4861 gaccaattga gatgtatgtc tgaattagtt gttttcaaag caaatgaact agcgattagt
     4921 cgctatgact taacggagca tgaaaccaag ctaattttat gctgtgtggc actactcaac
     4981 cccacgattg aaaaccctac aaggaaagaa cggacggtat cgttcactta taaccaatac
     5041 gttcagatga tgaacatcag tagggaaaat gcttatggtg tattagctaa agcaaccaga
     5101 gagctgatga cgagaactgt ggaaatcagg aatcctttgg ttaaaggctt tgagattttc
     5161 cagtggacaa actatgccaa gttctcaagc gaaaaattag aattagtttt tagtgaagag
     5221 atattgcctt atcttttcca gttaaaaaaa ttcataaaat ataatctgga acatgttaag
     5281 tcttttgaaa acaaatactc tatgaggatt tatgagtggt tattaaaaga actaacacaa
     5341 aagaaaactc acaaggcaaa tatagagatt agccttgatg aatttaagtt catgttaatg
     5401 cttgaaaata actaccatga gtttaaaagg cttaaccaat gggttttgaa accaataagt
     5461 aaagatttaa acacttacag caatatgaaa ttggtggttg ataagcgagg ccgcccgact
     5521 gatacgttga ttttccaagt tgaactagat agacaaatgg atctcgtaac cgaacttgag
     5581 aacaaccaga taaaaatgaa tggtgacaaa ataccaacaa ccattacatc agattcctac
     5641 ctacataacg gactaagaaa aacactacac gatgctttaa ctgcaaaaat tcagctcacc
     5701 agttttgagg caaaattttt gagtgacatg caaagtaagt atgatctcaa tggttcgttc
     5761 tcatggctca cgcaaaaaca acgaaccaca ctagagaaca tactggctaa atacggaagg
     5821 atctgaggtt cttatggctc ttgtatctat cagtgaagca tcaagactaa cacgcta
//
