LOCUS       sequence-399637-        6270 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Even ORI-marker screen destination vector with sfYFP dropout for 
            barcoded-ORI insertion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6270)
  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 6270)
  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_141
FEATURES             Location/Qualifiers
     source          1..6270
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(144..160)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(144..160)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(157..179)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      350..738
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(887..981)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(1002..1661)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1576..1595
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1662..1764)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     1851..1873
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1865..1882
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1866..1882
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     RBS             2182..2193
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2200..2913
                     /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"
     CDS             3388..4413
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3388..4413
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             5038..5751
                     /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
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     oriT            6152..6260
                     /note="incP origin of transfer"
ORIGIN
        1 atacttgaga ccaccccagg atacatagat taccacaact ccgagccctt ccacctagcg
       61 tgctaaccac gcacgctatt gttgtatagg ctaggtggag gctcagtgat gataagtctg
      121 cgatggtgga tgcatgtgtc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
      181 agagggcaca atcctattcc gcgctatccg acaatctcca agacattagg tggagttcag
      241 ttcggcgtat ggcatatgtc gctggaaaga acatgtgagc aaaaggccag caaaaggcca
      301 ggaaccgtaa aaaggccgcg ttgctggcgt tacagctggg cgcgccccat gtcagccgtt
      361 aagtgttcct gtgtcactca aaattgcttt gagaggctct aagggcttct cagtgcgtta
      421 catccctggc ttgttgtcca caaccgttaa accttaaagg ctttaaaagc cttatatatt
      481 cttttttttc ttataaaact taaaacctta gaggctattt aagttgctga tttatattaa
      541 ttttattgtt caaacatgag agcttagtac gtgaaacatg agagcttagt acgttagcca
      601 tgagagctta gtacgttagc catgagggtt tagttcgtta aacatgagag cttagtacgt
      661 taaacatgag agcttagtac gtgaaacatg agagcttagt acgtactatc aacaggttga
      721 actgctgatc ttcagatcgg cgcgccggcc ggccgaagat cctttgatct tttctacggg
      781 gtctgacgct ctattcaaca aagccgccgt cccgtcaagt cagcgtaaat gggtaggggg
      841 cttcaaatcg tccccccata cgatataagt tgttactagt gcttggattc tcaccaataa
      901 aaaacgcccg gcggcaaccg agcgttctga acaaatccag atggagttct gaggtcatta
      961 ctggatctat caacaggagt ccaagcgagc tcgatatcaa attacgcccc gccctgccac
     1021 tcatcgcagt actgttgtaa ttcattaagc attctgccga catggaagcc atcacaaacg
     1081 gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt ataatatttg
     1141 cccatggtga aaacgggggc gaagaagttg tccatattgg ccacgtttaa atcaaaactg
     1201 gtgaaactca cccagggatt ggctgagacg aaaaacatat tctcaataaa ccctttaggg
     1261 aaataggcca ggttttcacc gtaacacgcc acatcttgcg aatatatgtg tagaaactgc
     1321 cggaaatcgt cgtggtattc actccagagc gatgaaaacg tttcagtttg ctcatggaaa
     1381 acggtgtaac aagggtgaac actatcccat atcaccagct caccgtcttt cattgccata
     1441 cgaaattccg gatgagcatt catcaggcgg gcaagaatgt gaataaaggc cggataaaac
     1501 ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa tatccagctg aacggtctgg
     1561 ttataggtac attgagcaac tgactgaaat gcctcaaaat gttctttacg atgccattgg
     1621 gatatatcaa cggtggtata tccagtgatt tttttctcca ttttagcttc cttagctcct
     1681 gaaaatctcg ataactcaaa aaatacgccc ggtagtgatc ttatttcatt atggtgaaag
     1741 ttggaacctc ttacgtgccg atcaacgtct cattgatacc tgaaacaaaa cccatcgtac
     1801 ggccaaggaa gtctccaata actgtgatcc accacaagcg ccagggtttt cccagtcacg
     1861 acgttgtaaa acgacggcca gtcatgcata atccgcacgc atctggaata aggaagtgcc
     1921 attccgcctg accttagcgt gcgaacagca cgctattgtt gtatcattac tcgcatccat
     1981 tctcaggctg tctcgtctcg tctccgaggt ctcaggagaa tgtgaagagc ttcgaaagtg
     2041 aaacgtgatt tcatgcgtca ttttgaacat tttgtaaatc ttatttaata atgtgtgcgg
     2101 caattcacat ttaatttatg aatgttttct taacatcgcg gcaactcaag aaacggcagg
     2161 ttcggatctt agctactaga gaaagaggag aaatactaga tgcgtaaagg cgaagaactg
     2221 ttcactggtg tcgtccctat tctggtggaa ctggatggtg atgtcaacgg tcataagttt
     2281 tccgtgcgtg gcgagggtga aggtgacgca actaatggta aactgacgct gaagttcatc
     2341 tgtactactg gtaaactgcc ggtaccttgg ccgactctgg taacgacgct gacttatggt
     2401 gttcagtgct ttgctcgtta tccggaccat atgaagcagc atgacttctt caagtccgcc
     2461 atgccggaag gctatgtgca ggaacgcacg atttccttta aggatgacgg cacgtacaaa
     2521 acgcgtgcgg aagtgaaatt tgaaggcgat accctggtaa accgcattga gctgaaaggc
     2581 attgacttta aagaagacgg caatatcctg ggccataagc tggaatacaa ttttaacagc
     2641 cacaatgttt acatcaccgc cgataaacaa aaaaatggca ttaaagcgaa ttttaaaatt
     2701 cgccacaacg tggaggatgg cagcgtgcag ctggctgatc actaccagca aaacactcca
     2761 atcggtgatg gtcctgttct gctgccagac aatcactatc tgagctacca aagcgttctg
     2821 tctaaagatc cgaacgagaa acgcgatcat atggttctgc tggagttcgt aaccgcagcg
     2881 ggcatcacgc atggtatgga tgaactgtac aaataattca gccaaaaaac ttaagaccgc
     2941 cggtcttgtc cactaccttg cagtaatgcg gtggacagga tcggcggttt tcttttctct
     3001 tctcaagaag ctcttcagca aggagaactg atattgaagg agcatttttt gggcttggct
     3061 ggagctagtg gaggtcaaca atgaatgcct attttggttt agtcgtccag gcggtgagca
     3121 caaaatttgt gtcgtttgac aagatggttc atttaggcaa ctggtcagat cagccccact
     3181 tgtagcagta gcggcggcgc tcgaagtgtg actcttatta gcagacagga acgaggacat
     3241 tattatcatc tgctgcttgg tgcacgataa cttggtgcgt ttgtcaagca aggtaagtgg
     3301 acgacccggt cataccttct taagttcgcc cttcctccct ttatttcaga ttcaatctga
     3361 cttacctatt ctacccaagc atccaaaatg aaaaagcctg aactcaccgc gacgtctgtc
     3421 gagaagtttc tgatcgaaaa gttcgacagc gtgtccgacc tgatgcagct ctcggagggc
     3481 gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat
     3541 agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg
     3601 ctcccgattc cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc
     3661 tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt
     3721 ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc
     3781 gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata
     3841 tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt
     3901 gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc
     3961 cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata
     4021 acagcggtca ttgactggag cgaggcaatg ttcggggatt cccaatacga ggtcgccaac
     4081 atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg
     4141 aggcatccgg agcttgcagg atcgccacga ctccgggcgt atatgctccg cattggtctt
     4201 gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt
     4261 cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc
     4321 agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga
     4381 cgccccagca ctcgtccgag ggcaaaggaa tgagcttact taacgttact gaaatcatca
     4441 aacagcttga cgaatctgga tataagatcg ttggtgtcga tgtcagctcc ggagttgaga
     4501 caaatggtgt tcaggatctc gataagatac gttcatttgt ccaagcagca aagagtgcct
     4561 tctagtgatt taatagctcc atgtcaacaa gaataaaacg cgtttcgggt ttacctcttc
     4621 cagatacagc tcatctgcaa tgcatcgacc gtctgagaca tgcgctatac aggagaactg
     4681 atattgaagg agcatttttt gggcttggct ggagctagtg gaggtcaaca atgaatgcct
     4741 attttggttt agtcgtccag gcggtgagca caaaatttgt gtcgtttgac aagatggttc
     4801 atttaggcaa ctggtcagat cagccccact tgtagcagta gcggcggcgc tcgaagtgtg
     4861 actcttatta gcagacagga acgaggacat tattatcatc tgctgcttgg tgcacgataa
     4921 cttggtgcgt ttgtcaagca aggtaagtgg acgacccggt cataccttct taagttcgcc
     4981 cttcctccct ttatttcaga ttcaatctga cttacctatt ctacccaagc atccaaaatg
     5041 cgtaaaggcg aagaactgtt cactggtgtc gtccctattc tggtggaact ggatggtgat
     5101 gtcaacggtc ataagttttc cgtgcgtggc gagggtgaag gtgacgcaac taatggtaaa
     5161 ctgacgctga agttcatctg tactactggt aaactgccgg taccttggcc gactctggta
     5221 acgacgctga cttatggtgt tcagtgcttt gctcgttatc cggaccatat gaagcagcat
     5281 gacttcttca agtccgccat gccggaaggc tatgtgcagg aacgcacgat ttcctttaag
     5341 gatgacggca cgtacaaaac gcgtgcggaa gtgaaatttg aaggcgatac cctggtaaac
     5401 cgcattgagc tgaaaggcat tgactttaaa gaagatggca atatcctggg ccataagctg
     5461 gaatacaatt ttaacagcca caatgtttac atcaccgccg ataaacaaaa aaatggcatt
     5521 aaagcgaatt ttaaaattcg ccacaacgtg gaggatggca gcgtgcagct ggctgatcac
     5581 taccagcaaa acactccaat cggtgatggt cctgttctgc tgccagacaa tcactatctg
     5641 agcacgcaaa gcgttctgtc taaagatccg aacgagaaac gcgatcatat ggttctgctg
     5701 gagttcgtaa ccgcagcggg catcacgcat ggtatggatg aactgtacaa atgagcttac
     5761 ttaacgttac tgaaatcatc aaacagcttg acgaatctgg atataagatc gttggtgtcg
     5821 atgtcagctc cggagttgag acaaatggtg ttcaggatct cgataagata cgttcatttg
     5881 tccaagcagc aaagagtgcc ttctagtgat ttaatagctc catgtcaaca agaataaaac
     5941 gcgtttcggg tttacctctt ccagatacag ctcatctgca atgcatcgac cgtctgagac
     6001 atgcgctaca gaggagcttt tccgctgcat aaccctgctt cggggtcatt atagcgattt
     6061 tttcggtata tccatccttt ttcgcacgat atacaggatt ttgccaaagg gttcgtgtag
     6121 actttccttg gtgtatccaa cggcgtcagc cgggcaggat aggtgaagta ggcccacccg
     6181 cgagcgggtg ttccttcttc actgtccctt attcgcacct ggcggtgctc aacgggaatc
     6241 ctgctctgcg aggctggccg tacgctaggt
//
