LOCUS       sequence-398512-        5214 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 4136 to 4170)
  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 5214)
  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_136
FEATURES             Location/Qualifiers
     source          1..5214
                     /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             3145..3960
                     /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             3145..3960
                     /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     complement(3218..3237)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        4136..4170
                     /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            5096..5204
                     /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 aggagcaacc atcatcgatg aattgtgtct caaaatctct
     3061 gatgttacat tgcacaagat aaaaatatat catcatgaac aataaaactg tctgcttaca
     3121 taaacagtaa tacaaggggt gttaatgagc catattcaac gggaaacgtc ttgctcgagg
     3181 ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat
     3241 gtcgggcaat caggtgcgac aatctatcga ttgtatggga agcccgatgc gccagagttg
     3301 tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta
     3361 aactggctga cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat
     3421 gatgcatggt tactcaccac tgcgatcccc ggaaaaacag cattccaggt attagaagaa
     3481 tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat
     3541 tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg
     3601 caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc
     3661 tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc accggattca
     3721 gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata
     3781 ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta
     3841 tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt
     3901 attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa
     3961 gcttgagcgc atgctcgagt acttcgcctg gaccatgctc gccgtcgtct tcggcttcct
     4021 cctcttcgtc aacctcgcct acgtccccct cggccactgg gccgaggcct tcgccggctt
     4081 cttcaaattt tcagggctgc cgcaccccat cgactggggg ctcgctatac aggagttgac
     4141 ggctagctca gtcctaggta cagtgctagc tactaggagg aaaaaaatgg tgagtaaggg
     4201 tgaagccgtg attaaagagt tcatgcgctt caaagtgcat atggagggct caatgaacgg
     4261 ccatgaattt gaaattgaag gtgaaggaga aggccgtccg tatgagggca cccagaccgc
     4321 caaactgaaa gtcacaaagg gtggcccgct gccgttttcg tgggatattt tatcccctca
     4381 attcatgtac ggcagccggg cgtttattaa acacccggcg gatatcccgg attactataa
     4441 gcaatctttt ccagaaggct ttaaatggga gcgcgtgatg aattttgaag acggaggtgc
     4501 tgttaccgtg acccaggaca cgagcttaga agacggtacc ttgatctata aggtaaagct
     4561 gcgtggtacc aatttcccgc cggatgggcc agttatgcag aaaaagacta tgggctggga
     4621 agcatcgacg gagcgtcttt accccgaaga cggcgtcctg aaaggcgata ttaaaatggc
     4681 cctgcgactg aaagacggcg gtcgctacct tgcagatttc aaaacaacgt ataaagcgaa
     4741 aaaacctgtg cagatgcccg gggcgtacaa cgttgatcgc aaactagata tcacttcaca
     4801 caacgaagat tacacggtag tcgaacagta tgaacgtagc gaggggcgcc atagcaccgg
     4861 aggcatggat gaactctata aataagcttc gctcgtgctc gccgcgacct tcctcttcat
     4921 gcacctcttc ggcatcgggc tgcacaacgc tacagaggag cttttccgct gcataaccct
     4981 gcttcggggt cattatagcg attttttcgg tatatccatc ctttttcgca cgatatacag
     5041 gattttgcca aagggttcgt gtagactttc cttggtgtat ccaacggcgt cagccgggca
     5101 ggataggtga agtaggccca cccgcgagcg ggtgttcctt cttcactgtc ccttattcgc
     5161 acctggcggt gctcaacggg aatcctgctc tgcgaggctg gccgtacgct aggt
//
