LOCUS       sequence-398516-        5547 bp    DNA     circular SYN 15-APR-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 4469 to 4503)
  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 5547)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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_137
FEATURES             Location/Qualifiers
     source          1..5547
                     /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        join(3026..3031,3032..3048,3049..3054)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             3103..4293
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSITGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     complement(3455..3474)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4469..4503
                     /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            5429..5537
                     /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 aggagttgac agcttatcat cgataagctt taatgcggta
     3061 gtttatcaca gttaaattgc taacgcagtc aggcaccgtg taatgaaatc taacaatgcg
     3121 ctcatcgtca tcctcggcac cgtcaccctg gatgctgtag gcataggctt ggttatgccg
     3181 gtactgccgg gcctcttgcg ggatatcgtc cattccgaca gcatcgccag tcactatggc
     3241 gtgctgctag cgctatatgc gttgatgcaa tttctatgcg cacccgttct cggagcactg
     3301 tccgaccgct ttggccgccg cccagtcctg ctcgcttcgc tacttggagc cactatcgac
     3361 tacgcgatca tggcgaccac acccgtcctg tggattctct acgccggacg catcgtggcc
     3421 ggcatcaccg gcgccacagg tgcggttgct ggcgcctata tcgccgacat caccgatggg
     3481 gaagatcggg ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg tatggtggca
     3541 ggccccgtgg ccgggggact gttgggcgcc atctccctgc acgcaccatt ccttgcggcg
     3601 gcggtgctca acggcctcaa cctactactg ggctgcttcc taatgcagga gtcgcataag
     3661 ggagagcgcc gtccgatgcc cttgagagcc ttcaacccag tcagctcctt ccggtgggcg
     3721 cggggcatga ccattgtggc cgcacttatg actgtcttct ttatcatgca actcgtagga
     3781 caggtgccgg cagcgctctg ggtcattttc ggcgaggacc gctttcgctg gagcgcgacg
     3841 atgatcggcc tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca agccttcgtc
     3901 actggtcccg ccaccaaacg tttcggcgag aagcaggcca ttatcgccgg catggcggcc
     3961 gacgcgctgg gctacgtctt gctggcgttc gcgacgcgag gctggatggc cttccccatt
     4021 atgattcttc tcgcttccgg cggcatcggg atgcccgcgt tgcaggccat gctgtccagg
     4081 caggtagatg acgaccatca gggacagctt caaggatcgc tcgcggctct taccagccta
     4141 acttcgatca ctggaccgct gatcgtcacg gcgatttatg ccgcctcggc gagcacatgg
     4201 aacgggttgg catggattgt aggcgccgcc ctataccttg tctgcctccc cgcgttgcgt
     4261 cgcggtgcat ggagccgggc cacctcgacc tgagcttgag cgcatgctcg agtacttcgc
     4321 ctggaccatg ctcgccgtcg tcttcggctt cctcctcttc gtcaacctcg cctacgtccc
     4381 cctcggccac tgggccgagg ccttcgccgg cttcttcaaa ttttcagggc tgccgcaccc
     4441 catcgactgg gggctcgcta tacaggagtt gacggctagc tcagtcctag gtacagtgct
     4501 agctactagg aggaaaaaaa tggtgagtaa gggtgaagcc gtgattaaag agttcatgcg
     4561 cttcaaagtg catatggagg gctcaatgaa cggccatgaa tttgaaattg aaggtgaagg
     4621 agaaggccgt ccgtatgagg gcacccagac cgccaaactg aaagtcacaa agggtggccc
     4681 gctgccgttt tcgtgggata ttttatcccc tcaattcatg tacggcagcc gggcgtttat
     4741 taaacacccg gcggatatcc cggattacta taagcaatct tttccagaag gctttaaatg
     4801 ggagcgcgtg atgaattttg aagacggagg tgctgttacc gtgacccagg acacgagctt
     4861 agaagacggt accttgatct ataaggtaaa gctgcgtggt accaatttcc cgccggatgg
     4921 gccagttatg cagaaaaaga ctatgggctg ggaagcatcg acggagcgtc tttaccccga
     4981 agacggcgtc ctgaaaggcg atattaaaat ggccctgcga ctgaaagacg gcggtcgcta
     5041 ccttgcagat ttcaaaacaa cgtataaagc gaaaaaacct gtgcagatgc ccggggcgta
     5101 caacgttgat cgcaaactag atatcacttc acacaacgaa gattacacgg tagtcgaaca
     5161 gtatgaacgt agcgaggggc gccatagcac cggaggcatg gatgaactct ataaataagc
     5221 ttcgctcgtg ctcgccgcga ccttcctctt catgcacctc ttcggcatcg ggctgcacaa
     5281 cgctacagag gagcttttcc gctgcataac cctgcttcgg ggtcattata gcgatttttt
     5341 cggtatatcc atcctttttc gcacgatata caggattttg ccaaagggtt cgtgtagact
     5401 ttccttggtg tatccaacgg cgtcagccgg gcaggatagg tgaagtaggc ccacccgcga
     5461 gcgggtgttc cttcttcact gtcccttatt cgcacctggc ggtgctcaac gggaatcctg
     5521 ctctgcgagg ctggccgtac gctaggt
//
