LOCUS       sequence-398509-        5161 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 4083 to 4117)
  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 5161)
  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_135
FEATURES             Location/Qualifiers
     source          1..5161
                     /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"
     promoter        3068..3159
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3161..3229,3230..4021)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     terminator      4029..4068
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     promoter        4083..4117
                     /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            5043..5151
                     /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 aggagtagta gcccgcctaa tgagcgggct tttttttaat
     3061 tcccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     3121 ccctgataaa tgcttcaata atattgaaaa aggaagagta atgagcattc agcattttcg
     3181 tgtggcgctg attccgtttt ttgcggcgtt ttgcctgccg gtgtttgcgc atccggaaac
     3241 cctggtgaaa gtgaaagatg cggaagatca actgggtgcg cgcgtgggct atattgaact
     3301 ggatctgaac agcggcaaaa ttctggaatc ttttcgtccg gaagaacgtt ttccgatgat
     3361 gagcaccttt aaagtgctgc tgtgcggtgc ggttctgagc cgtgtggatg cgggccagga
     3421 acaactgggc cgtcgtattc attatagcca gaacgatctg gtggaatata gcccggtgac
     3481 cgaaaaacat ctgaccgatg gcatgaccgt gcgtgaactg tgcagcgcgg cgattaccat
     3541 gagcgataac accgcggcga acctgctgct gacgaccatt ggcggtccga aagaactgac
     3601 cgcgtttctg cataacatgg gcgatcatgt gacccgtctg gatcgttggg aaccggaact
     3661 gaacgaagcg attccgaacg atgaacgtga taccaccatg ccggcagcaa tggcgaccac
     3721 cctgcgtaaa ctgctgacgg gtgagctgct gaccctggca agccgccagc aactgattga
     3781 ttggatggaa gcggataaag tggcgggtcc gctgctgcgt agcgcgctgc cggctggctg
     3841 gtttattgcg gataaaagcg gtgcgggcga acgtggcagc cgtggcatta ttgcggcgct
     3901 gggcccggat ggtaaaccga gccgtattgt ggtgatttat accaccggca gccaggcgac
     3961 gatggatgaa cgtaaccgtc agattgcgga aattggcgcg agcctgatta aacattggta
     4021 agcttaccga tacaattaaa ggctcctttt ggagcctttt tttttggacc gctatacagg
     4081 agttgacggc tagctcagtc ctaggtacag tgctagctac taggaggaaa aaaatggtga
     4141 gtaagggtga agccgtgatt aaagagttca tgcgcttcaa agtgcatatg gagggctcaa
     4201 tgaacggcca tgaatttgaa attgaaggtg aaggagaagg ccgtccgtat gagggcaccc
     4261 agaccgccaa actgaaagtc acaaagggtg gcccgctgcc gttttcgtgg gatattttat
     4321 cccctcaatt catgtacggc agccgggcgt ttattaaaca cccggcggat atcccggatt
     4381 actataagca atcttttcca gaaggcttta aatgggagcg cgtgatgaat tttgaagacg
     4441 gaggtgctgt taccgtgacc caggacacga gcttagaaga cggtaccttg atctataagg
     4501 taaagctgcg tggtaccaat ttcccgccgg atgggccagt tatgcagaaa aagactatgg
     4561 gctgggaagc atcgacggag cgtctttacc ccgaagacgg cgtcctgaaa ggcgatatta
     4621 aaatggccct gcgactgaaa gacggcggtc gctaccttgc agatttcaaa acaacgtata
     4681 aagcgaaaaa acctgtgcag atgcccgggg cgtacaacgt tgatcgcaaa ctagatatca
     4741 cttcacacaa cgaagattac acggtagtcg aacagtatga acgtagcgag gggcgccata
     4801 gcaccggagg catggatgaa ctctataaat aagcttcgct cgtgctcgcc gcgaccttcc
     4861 tcttcatgca cctcttcggc atcgggctgc acaacgctac agaggagctt ttccgctgca
     4921 taaccctgct tcggggtcat tatagcgatt ttttcggtat atccatcctt tttcgcacga
     4981 tatacaggat tttgccaaag ggttcgtgta gactttcctt ggtgtatcca acggcgtcag
     5041 ccgggcagga taggtgaagt aggcccaccc gcgagcgggt gttccttctt cactgtccct
     5101 tattcgcacc tggcggtgct caacgggaat cctgctctgc gaggctggcc gtacgctagg
     5161 t
//
