LOCUS       sequence-397966-        3188 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 1 plasmid, methylated YFP dropout.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3188)
  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 3188)
  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: pGLDO_1 [alpha-sfYFP-beta]
FEATURES             Location/Qualifiers
     source          1..3188
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(487..1302)
                     /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(487..1302)
                     /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     1210..1229
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1294..1313
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     1423..1439
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1423..1439
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     terminator      1440..1486
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     1600..1622
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1614..1631
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1615..1631
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     RBS             1857..1868
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1875..2588
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(2763..2779)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2763..2779)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2776..2798)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(join(2951..3188,1..351))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3031..3050)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
       61 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
      121 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
      181 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
      241 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
      301 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
      361 ttgatctttt ctacggggtc tgacgctcta ttcaacaaag ccgccgtccc gtcaagtcag
      421 cgtaaatggg tagggggctt caaatcgtcc gctctgccag tgttacaacc aattaacaaa
      481 ttctgattag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
      541 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
      601 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
      661 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
      721 gacgactgaa tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac
      781 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
      841 tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg
      901 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
      961 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca
     1021 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     1081 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     1141 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg
     1201 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     1261 tcgcggcctc gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact
     1321 gtttatgtaa gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta
     1381 acatcagaga ttttgagaca caacgtggct ttcccccgcc gctctagaac tagtggatcc
     1441 aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgc
     1501 attatacgag acgtccaggt tgggatacct gaaacaaaac ccatcgtacg gccaaggaag
     1561 tctccaataa ctgtgatcca ccacaagcgc cagggttttc ccagtcacga cgttgtaaaa
     1621 cgacggccag tcatgcataa tccgcacgca tctggaataa ggaagtgcca ttccgcctga
     1681 cctgacgctc ttcaatgtga agagcttcga aagtgaaacg tgatttcatg cgtcattttg
     1741 aacattttgt aaatcttatt taataatgtg tgcggcaatt cacatttaat ttatgaatgt
     1801 tttcttaaca tcgcggcaac tcaagaaacg gcaggttcgg atcttagcta ctagagaaag
     1861 aggagaaata ctagatgcgt aaaggcgaag aactgttcac tggtgtcgtc cctattctgg
     1921 tggaactgga tggtgatgtc aacggtcata agttttccgt gcgtggcgag ggtgaaggtg
     1981 acgcaactaa tggtaaactg acgctgaagt tcatctgtac tactggtaaa ctgccggtac
     2041 cttggccgac tctggtaacg acgctgactt atggtgttca gtgctttgct cgttatccgg
     2101 accatatgaa gcagcatgac ttcttcaagt ccgccatgcc ggaaggctat gtgcaggaac
     2161 gcacgatttc ctttaaggat gacggcacgt acaaaacgcg tgcggaagtg aaatttgaag
     2221 gcgataccct ggtaaaccgc attgagctga aaggcattga ctttaaagaa gacggcaata
     2281 tcctgggcca taagctggaa tacaatttta acagccacaa tgtttacatc accgccgata
     2341 aacaaaaaaa tggcattaaa gcgaatttta aaattcgcca caacgtggag gatggcagcg
     2401 tgcagctggc tgatcactac cagcaaaaca ctccaatcgg tgatggtcct gttctgctgc
     2461 cagacaatca ctatctgagc taccaaagcg ttctgtctaa agatccgaac gagaaacgcg
     2521 atcatatggt tctgctggag ttcgtaaccg cagcgggcat cacgcatggt atggatgaac
     2581 tgtacaaata attcagccaa aaaacttaag accgccggtc ttgtccacta ccttgcagta
     2641 atgcggtgga caggatcggc ggttttcttt tctcttctca agaagctctt cagcatgaag
     2701 agcgcaaggc taggtggagg ctcagtgatg ataagtctgc gatggtggat gcatgtgtca
     2761 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca gagggcacaa tcctattccg
     2821 cgctatccga caatctccaa gacattaggt ggagttcagt tcggcgtatg gcatatgtcg
     2881 ctggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     2941 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     3001 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     3061 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     3121 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     3181 tcgttcgc
//
