LOCUS       sequence-398535-        6352 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 1061 to 1095)
  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 6352)
  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_166
FEATURES             Location/Qualifiers
     source          1..6352
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(134..139,140..156,157..162)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             352..885
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        1061..1095
                     /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            2021..2129
                     /note="incP origin of transfer"
     primer_bind     complement(2283..2299)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2283..2299)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2296..2318)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      2489..2877
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(3026..3120)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(3141..3800)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3715..3734
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3801..3903)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     3990..4012
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4004..4021
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4005..4021
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(4301..5449)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     IVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     rep_origin      join(5842..6352,1..21)
                     /label=oriV
                     /note="incP origin of replication"
ORIGIN
        1 gtatccacaa cgccggcggc cctacatggc tctgctgtag tgagtgggtt gcgctccggc
       61 agcggtcctg atcccccgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
      121 cgctagcaag gagttgacat aagcctgttc ggttcgtaaa ctgtaatgca agtagcgtat
      181 gcgctcacgc aactggtcca gaaccttgac cgaacgcagc ggtggtaacg gcgcagtggc
      241 ggttttcatg gcttgttatg actgtttttt tgtacagtct atgcctcggg catccaagca
      301 gcaagcgcgt tacgccgtgg gtcgatgttt gatgttatgg agcagcaacg aatgttacgc
      361 agcagcaacg atgttacgca gcagggcagt cgccctaaaa caaagttagg tggctcaagt
      421 atgggcatca ttcgcacatg taggctcggc cctgaccaag tcaaatccat gcgggctgct
      481 cttgatcttt tcggtcgtga gttcggagac gtagccacct actcccaaca tcagccggac
      541 tccgattacc tcgggaactt gctccgtagt aagacattca tcgcgcttgc tgccttcgac
      601 caagaagcgg ttgttggcgc tctcgcggct tacgttctgc ccaagtttga gcagccgcgt
      661 agtgagatct atatctatga tctcgcagtc tccggagagc accggaggca gggcattgcc
      721 accgcgctca tcaatctcct caagcatgag gccaacgcgc ttggtgctta tgtgatctac
      781 gtgcaagcag attacggtga cgatcccgca gtggctctct atacaaagtt gggcatacgg
      841 gaagaagtga tgcactttga tatcgaccca agtaccgcca cctaagcttg agcgcatgct
      901 cgagtacttc gcctggacca tgctcgccgt cgtcttcggc ttcctcctct tcgtcaacct
      961 cgcctacgtc cccctcggcc actgggccga ggccttcgcc ggcttcttca aattttcagg
     1021 gctgccgcac cccatcgact gggggctcgc tatacaggag ttgacggcta gctcagtcct
     1081 aggtacagtg ctagctacta ggaggaaaaa aatggtgagt aagggtgaag ccgtgattaa
     1141 agagttcatg cgcttcaaag tgcatatgga gggctcaatg aacggccatg aatttgaaat
     1201 tgaaggtgaa ggagaaggcc gtccgtatga gggcacccag accgccaaac tgaaagtcac
     1261 aaagggtggc ccgctgccgt tttcgtggga tattttatcc cctcaattca tgtacggcag
     1321 ccgggcgttt attaaacacc cggcggatat cccggattac tataagcaat cttttccaga
     1381 aggctttaaa tgggagcgcg tgatgaattt tgaagacgga ggtgctgtta ccgtgaccca
     1441 ggacacgagc ttagaagacg gtaccttgat ctataaggta aagctgcgtg gtaccaattt
     1501 cccgccggat gggccagtta tgcagaaaaa gactatgggc tgggaagcat cgacggagcg
     1561 tctttacccc gaagacggcg tcctgaaagg cgatattaaa atggccctgc gactgaaaga
     1621 cggcggtcgc taccttgcag atttcaaaac aacgtataaa gcgaaaaaac ctgtgcagat
     1681 gcccggggcg tacaacgttg atcgcaaact agatatcact tcacacaacg aagattacac
     1741 ggtagtcgaa cagtatgaac gtagcgaggg gcgccatagc accggaggca tggatgaact
     1801 ctataaataa gcttcgctcg tgctcgccgc gaccttcctc ttcatgcacc tcttcggcat
     1861 cgggctgcac aacgctacag aggagctttt ccgctgcata accctgcttc ggggtcatta
     1921 tagcgatttt ttcggtatat ccatcctttt tcgcacgata tacaggattt tgccaaaggg
     1981 ttcgtgtaga ctttccttgg tgtatccaac ggcgtcagcc gggcaggata ggtgaagtag
     2041 gcccacccgc gagcgggtgt tccttcttca ctgtccctta ttcgcacctg gcggtgctca
     2101 acgggaatcc tgctctgcga ggctggccgt acgctaggta tacttgagac caccccagga
     2161 tacatagatt accacaactc cgagcccttc cacctagcgt gctaaccacg cacgctattg
     2221 ttgtataggc taggtggagg ctcagtgatg ataagtctgc gatggtggat gcatgtgtca
     2281 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca gagggcacaa tcctattccg
     2341 cgctatccga caatctccaa gacattaggt ggagttcagt tcggcgtatg gcatatgtcg
     2401 ctggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     2461 tgctggcgtt acagctgggc gcgccccatg tcagccgtta agtgttcctg tgtcactcaa
     2521 aattgctttg agaggctcta agggcttctc agtgcgttac atccctggct tgttgtccac
     2581 aaccgttaaa ccttaaaggc tttaaaagcc ttatatattc ttttttttct tataaaactt
     2641 aaaaccttag aggctattta agttgctgat ttatattaat tttattgttc aaacatgaga
     2701 gcttagtacg tgaaacatga gagcttagta cgttagccat gagagcttag tacgttagcc
     2761 atgagggttt agttcgttaa acatgagagc ttagtacgtt aaacatgaga gcttagtacg
     2821 tgaaacatga gagcttagta cgtactatca acaggttgaa ctgctgatct tcagatcggc
     2881 gcgccggccg gccgaagatc ctttgatctt ttctacgggg tctgacgctc tattcaacaa
     2941 agccgccgtc ccgtcaagtc agcgtaaatg ggtagggggc ttcaaatcgt ccccccatac
     3001 gatataagtt gttactagtg cttggattct caccaataaa aaacgcccgg cggcaaccga
     3061 gcgttctgaa caaatccaga tggagttctg aggtcattac tggatctatc aacaggagtc
     3121 caagcgagct cgatatcaaa ttacgccccg ccctgccact catcgcagta ctgttgtaat
     3181 tcattaagca ttctgccgac atggaagcca tcacaaacgg catgatgaac ctgaatcgcc
     3241 agcggcatca gcaccttgtc gccttgcgta taatatttgc ccatggtgaa aacgggggcg
     3301 aagaagttgt ccatattggc cacgtttaaa tcaaaactgg tgaaactcac ccagggattg
     3361 gctgagacga aaaacatatt ctcaataaac cctttaggga aataggccag gttttcaccg
     3421 taacacgcca catcttgcga atatatgtgt agaaactgcc ggaaatcgtc gtggtattca
     3481 ctccagagcg atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca agggtgaaca
     3541 ctatcccata tcaccagctc accgtctttc attgccatac gaaattccgg atgagcattc
     3601 atcaggcggg caagaatgtg aataaaggcc ggataaaact tgtgcttatt tttctttacg
     3661 gtctttaaaa aggccgtaat atccagctga acggtctggt tataggtaca ttgagcaact
     3721 gactgaaatg cctcaaaatg ttctttacga tgccattggg atatatcaac ggtggtatat
     3781 ccagtgattt ttttctccat tttagcttcc ttagctcctg aaaatctcga taactcaaaa
     3841 aatacgcccg gtagtgatct tatttcatta tggtgaaagt tggaacctct tacgtgccga
     3901 tcaacgtctc attgatacct gaaacaaaac ccatcgtacg gccaaggaag tctccaataa
     3961 ctgtgatcca ccacaagcgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag
     4021 tcatgcataa tccgcacgca tctggaataa ggaagtgcca ttccgcctga ccttagcgtg
     4081 cgaacagcac gctattgttg tatcattact cgcatccatt ctcaggctgt ctcgtctcgt
     4141 ctccgaggtc tcaggagaat gagtcagggt cacgcgtagg acgcgtcgag tagggttaga
     4201 ttcccttgac ttgcgtgttc cgcagccaca cgccgtgtga agctggggag gcgatgcagg
     4261 tggctgctga acccccagcc ggaactgacc ccacaaggcc ctagcgtttg caatgcacca
     4321 ggtcatcatt gacccaggcg tgttccacca ggccgctgcc tcgcaactct tcgcaggctt
     4381 cgccgacctg ctcgcgccac ttcttcacgc gggtggaatc cgatccgcac atgaggcgga
     4441 aggtttccag cttgagcggg tacggctccc ggtgcgagct gaaatagtcg aacatccgtc
     4501 gggccgtcgg cgacagcttg cggtacttct cccatatgaa tttcgtgtag tggtcgccag
     4561 caaacagcac gacgatttcc tcgtcgatca ggacctggca acgggacgtt ttcttgccac
     4621 ggtccaggac gcggaagcgg tgcagcagcg acaccgattc caggtgccca acgcggtcgg
     4681 acgtgaagcc catcgccgtc gcctgtaggc gcgacaggca ttcctcggcc ttcgtgtaat
     4741 accggccatt gatcgaccag cccaggtcct ggcaaagctc gtagaacgtg aaggtgatcg
     4801 gctcgccgat aggggtgcgc ttcgcgtact ccaacacctg ctgccacacc agttcgtcat
     4861 cgtcggcccg cagctcgacg ccggtgtagg tgatcttcac gtccttgttg acgtggaaaa
     4921 tgaccttgtt ttgcagcgcc tcgcgcggga ttttcttgtt gcgcgtggtg aacagggcag
     4981 agcgggccgt gtcgtttggc atcgctcgca tcgtgtccgg ccacggcgca atatcgaaca
     5041 aggaaagctg catttccttg atctgctgct tcgtgtgttt cagcaacgcg gcctgcttgg
     5101 cttcgctgac ctgttttgcc aggtcctcgc cggcggtttt tcgcttcttg gtcgtcatag
     5161 ttcctcgcgt gtcgatggtc atcgacttcg ccaaacctgc cgcctcctgt tcgagacgac
     5221 gcgaacgctc cacggcggcc gatggcgcgg gcagggcagg gggagccagt tgcacgctgt
     5281 cgcgctcgat cttggccgta gcttgctgga ctatcgagcc gacggactgg aaggtttcgc
     5341 ggggcgcacg catgacggtg cggcttgcga tggtttcggc atcctcggcg gaaaaccccg
     5401 cgtcgatcag ttcttgcctg tatgccttcc ggtcaaacgt ccgattcatt caccctcctt
     5461 gcgggattgc cccggaatta attccccgga tcgatccgtc gatcttgatc ccctgcgcca
     5521 tcagatcctt ggcggcaaga aagccatcca gtttactttg cagggcttcc caaccttacc
     5581 agagggcgcc ccagctggca attccggttc gcttgctgtc cataaaaccg cccagtctag
     5641 ctatcgccat gtaagcccac tgcaagctac ctgctttctc tttgcgcttg cgttttccct
     5701 tgtccagata gcccagtagc tgacattcat ccggggtcag caccgtttct gcggactggc
     5761 tttctacgtg gctgccattt ttggggtgag gccgttcgcg gccgaggggc gcagcccctg
     5821 gggggatggg aggcccgcgt tagcgggccg ggagggttcg agaagggggg gcacccccct
     5881 tcggcgtgcg cggtcacgcg cacagggcgc agccctggtt aaaaacaagg tttataaata
     5941 ttggtttaaa agcaggttaa aagacaggtt agcggtggcc gaaaaacggg cggaaaccct
     6001 tgcaaatgct ggattttctg cctgtggaca gcccctcaaa tgtcaatagg tgcgcccctc
     6061 atctgtcagc actctgcccc tcaagtgtca aggatcgcgc ccctcatctg tcagtagtcg
     6121 cgcccctcaa gtgtcaatac cgcagggcac ttatccccag gcttgtccac atcatctgtg
     6181 ggaaactcgc gtaaaatcag gcgttttcgc cgatttgcga ggctggccag ctccacgtcg
     6241 ccggccgaaa tcgagcctgc ccctcatctg tcaacgccgc gccgggtgag tcggcccctc
     6301 aagtgtcaac gtccgcccct catctgtcag tgagggccaa gttttccgcg ag
//
