LOCUS       sequence-104118-        9493 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To construct C-terminal GFP fusions, single crossover integration 
            vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9493)
  AUTHORS   Eberhardt A, Wu LJ, Errington J, Vollmer W, Veening JW
  TITLE     Cellular localization of choline-utilization proteins in 
            Streptococcus pneumoniae using novel fluorescent reporter systems.
  JOURNAL   Mol Microbiol. 2009 Oct;74(2):395-408. doi: 
            10.1111/j.1365-2958.2009.06872.x. Epub 2009 Sep 8.
  PUBMED    19737355
REFERENCE   2  (bases 1 to 9493)
  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"; doi: 
            "10.1111/j.1365-2958.2009.06872.x"; journalName: "Mol Microbiol"; 
            date: "2009-10"; volume: "74"; issue: "2"; pages: "395-408"
COMMENT     Sequence Label: pAE03
FEATURES             Location/Qualifiers
     source          1..9493
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(20..38)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        106..210
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(211..279,280..1071)
                     /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"
     primer_bind     complement(429..448)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1242..1830
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1731..1750
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    7609..7630
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7645..7650,7651..7668,7669..7675)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7683..7699
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     7688..7710
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7707..7723
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7707..7723
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_feature    7736..7792
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     CDS             join(7801..7803,7804..8517)
                     /codon_start=1
                     /product="Cycle 3 GFP (Crameri et al., 1996)"
                     /label=Cycle 3 GFP
                     /note="mammalian codon-optimized"
                     /translation="ASKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(7831..7859)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     8454..8474
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
ORIGIN
        1 aattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
       61 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
      121 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
      181 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
      241 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
      301 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
      361 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
      421 aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt
      481 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
      541 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
      601 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
      661 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
      721 ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa
      781 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
      841 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
      901 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
      961 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     1021 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     1081 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     1141 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     1201 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     1261 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     1321 gatcaagagc tatcaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     1381 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     1441 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     1501 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     1561 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     1621 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     1681 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     1741 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     1801 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcccg acctcgagct
     1861 ggatacttcc cgtccgccag ggggacatgc cggcgatgct gaaggtcgcg cgcattcccg
     1921 atgaagaggc cggttaccgc ctgtttgagg atatagtaat ctttctaaat agctttggat
     1981 tggaggagta tggccactaa tactaagttc agctaataaa aaaatttgct aaagaactcc
     2041 agaaaagtaa gcacctgtta ttgcaataaa attagcctaa ttgagagaag tttctataga
     2101 atttttcata tacttaacga gtgctttcac ctttgaatat agtccttccc acttatcatc
     2161 acactctccc cgatagcctt ttctagctat atccagtaaa gttacatgct ctttaggtaa
     2221 aagaggtata gcccattctg cagcgacatc tttcgaggta atttcaccag tagtcactgt
     2281 ttgccacatt cgagctaggg ttaaaattac attacgctca tcacctttta tcccctcaat
     2341 tagttctggc aaagaatcct taattgctct tcgaatatct gtcaaaggta cggagacaag
     2401 tatacttgaa gaatcaggac caaatagaga aatactattc tttcttgctt gtgctaaaac
     2461 aatagccaaa tcaggatcat agcttggttc ctgaatttgt ccattctcaa attcacccct
     2521 gagccactca ccgtatataa attctctttt tggaggatat tgccaaggga caacttcact
     2581 cctatttata accgtaactt caagtggtct aacagaatcc gtatttccaa tctttcctga
     2641 tatagtcatt agtctttctg ttagtttttt tcgagttaat tgaggtaaac tatgattcac
     2701 gacgactaga acatctacat cgctgttaat gcgtaaacca ccatttactg ctgaaccaaa
     2761 tagatatact ccaactattg aacttccaaa taaatctttt acgattttta atgtttgaat
     2821 cgcttgattt ggtatttttc cgttaatcaa attgctcatg atttcacctc gttgattatg
     2881 ttcatataaa gtttatattg atactcaatt tacttaccct agattggaca tatacttaaa
     2941 ttactgttca ataaagctga ccgttagcgt ttaagtacat cctttcacaa tttgtctaca
     3001 gattaataat tattctttat tatacagatc gatcctctag acctaggcct taagatctga
     3061 tcatatgcat ccgcgggccc gggttaacgc gtaatccatg gatcaagaga caggatgagg
     3121 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggtgc cctgaatgaa
     3181 ctgcagaaag agctggtagt tggcgcactg ttcgaagaac tgccgatgtc cagtaagatt
     3241 cttactatgc tggttgaacc ggatgctggt aaagctactt gggttgctgc ttctacttat
     3301 ggtaccgata caactactgg tgaggaagtt aaaggagctc ttaaagaaat ccacttcagt
     3361 aggtggcccg gctccatgca ccgcgacgca acgcggggag gcagacaagg tatagggcgg
     3421 cgcctacaat ccatgccaac ccgttccatg tgctcgccga ggcggcataa atcgccgtga
     3481 cgatcagcgg tccagtgatc gaagttaggc tggtaagagc cgcgagcgat ccttgaagct
     3541 gtccctgatg gtcgtcatct acctgcctgg acagcatggc ctgcaacgcg ggcatcccga
     3601 tgccgccgga agcgagaaga atcataatgg ggaaggccat ccagcctcgc gtcgcgacta
     3661 agaaaatgcc gtcaaatccg ctcgccatga cttcactaac gatgcctttg aaaatcttca
     3721 agttcttttc tactaattca aggcgtgtct caccaggttt ttggtttgct ccggcgcaaa
     3781 tgcagacaat atcagcatcc ttgcagggta tgtttctctt tgatgtcttt ttgtttgtga
     3841 agtatttcac atttatattg tgcaacactt cacaaacttt tgcaagagaa aagttttgtc
     3901 tgatttatga acaaaaaaga aaccatcatt gatggtttct ttcggtaagt cccgtctagc
     3961 cttgccctca atggggaaga gaaccgctta agcccgagtc attatataaa ccatttagca
     4021 cgtaatcaaa gccaggctga ttctgaccgg gcacttgggc gctgccatta ttaaaaatca
     4081 cttttgcgtt ggttgtatcc gtgtccgcag gcagcgtcag cgtgtaaatt ccgtctgcat
     4141 ttttagtcat tggttttcca ggccaagatc cggtcaattc aattactcgg ctcccatcat
     4201 gtttatagat ataagcattt acctggctcc aatgattcgg attttgatag ccgatggttt
     4261 tggccgacgc tggatctctt ttaacaaaac tgtatttctc ggtcctcgtt acaccatcac
     4321 tgttcgttcc ttttaacatg atggtgtatg ttttgccaaa tggatctcct tttccgattg
     4381 tgaattgatc tccatcctta aacgccgtct ctggtccatt attgatttga taaacggctt
     4441 ttgttgtatt cgcatctgca cgcaaggtaa tcgtcagttg atcattgaaa gaatgtgtta
     4501 cacctgtttt gtaattctca aggaaaacat gaggcgcttt tgcaatatca tcaggataaa
     4561 gcacagctac agacctggca ttgatcgtgc ctgtcagttt accatcgttc acttgaaatg
     4621 aacccgctcc agctttattg tcatacctgc catcaggcaa ttttgttgcc gtattgatag
     4681 agacagagga tgaacctgca tttgccagca caacgccatg tgagccgcgc tgattcataa
     4741 atatctggtt gtttccattc gggttcgaga gttcctcagg ctgtccagcc atcacattgt
     4801 gaaatctatt gaccgcagtg atagcctgat cttcaaataa agcactcccg cgatcgccta
     4861 tttggctttt ccccgggaac ctcacaccat ttccgcctcc ctcaggtctg gaaaagaaaa
     4921 gaggcgtact gcctgaacga gaagctatca ccgcccagcc taaacggata tcatcatcgc
     4981 tcatccatgt cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     5041 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa
     5101 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tgaatcaggc
     5161 tgccgatacg gcgaaagcga tgatccatga aatgcgtcgt gagatgtcac gctatacgtg
     5221 gaaatccgcg atttatcttg tactgacgat attcttcgtc ctggcttcct gcgtgacggc
     5281 atttacgtgg tttatgaatg acgggtatag ccagattgct gaaatgcaga ggatggaagc
     5341 ggtctggcaa aagaaagccc cgctggctga catatcgcgc tgtgacggaa agccctgcgt
     5401 taaggtggac actcgcacta cgtatggtga taaagagaat acctggatga ttattaagaa
     5461 ataacatcat agagcacaga acgctgtgcg tcgatctccg gcggagaaag cattaattaa
     5521 acgtgtcggg gggatctgat aaccccagca aataaaggac attaaggata tggggtctga
     5581 aggccaatag aacgaaaacg tacgttagtg aagtaactgt ctgatatatc gaaacataat
     5641 gtacattgga aaacgccatc aaaacggtgt ctttttaatc gaaaattggc acttaacgga
     5701 ctttcttgtc tactaatcga tacaaattcc ccgtaggcgc tagggacctc tttagctcct
     5761 tggaagctgt cagtagtata cctaataatt tatctacatt ccctttagta acgtgtaact
     5821 ttccaaattt acaaaagcga ctcatagaat tatttcctcc cgttaaataa tagataacta
     5881 ttaaaaatag acaatacttg ctcataagta acggtactta aattgtttac tttggcgtgt
     5941 ttcattgctt gatgaaactg atttttagta aacagttgac gatattctcg attgacccat
     6001 tttgaaacaa agtacgtata tagcttccaa tatttatctg gaacatctgt ggtatggcgg
     6061 gtaagtttta ttaagacact gtttactttt ggtttaggat gaaagcattc cgctggcagc
     6121 ttaagcaatt gctgaatcga gacttgagtg tgcaagagca accctagtgt tcggtgaata
     6181 tccaaggtac gcttgtagaa tccttcttca acaatcagat agatgtcaga cgcatggctt
     6241 tcaaaaacca cttttttaat aatttgtgtg cttaaatggt aaggaatact cccaacaatt
     6301 ttatacctct gtttgttagg gaattgaaac tgtagaatat cttggtgaat taaagtgaca
     6361 cgagtattca gttttaattt ttctgacgat aagttgaata gatgactgtc taattcaata
     6421 gacgttacct gtttacttat tttagccagt ttcgtcgtta aatgcccttt acctgttcca
     6481 atttcgtaaa cggtatcggt ttcttttaaa ttcaattgtt ttattatttg gttgagtact
     6541 ttttcactcg ttaaaaagtt ttgagaatat tttatatttt tgttcatgta atcactcctt
     6601 cttaattaca aatttttagc atctaattta acttcaattc ctattataca aaattttaag
     6661 atactgcact atcaacacac tcttaagttt gcttctaagt cttatttcca taacttcttt
     6721 tacgtttccg ccattctttg ctgtttcgat ttttatgata tggtgcaagt cagcacgaac
     6781 acgaaccgtc ttatctccca ttatatcttt ttttgcactg attggtgtat catttcgttt
     6841 ttctttttgt gcgcctaaat ttcccacaat cactcacttc tttctatttc ttcttattct
     6901 tattttatca tcaacaatca caaatcactt gtgatttgtg ataagtgatt tgtgattaat
     6961 atataaaagc cctctttaaa gggcttttat gtttattttg agaaagatat aaaatcaata
     7021 tatccctttt ctccgatttt tacaacggca ttgtaggact ttctatcttt cgttttgatt
     7081 ccttttacca gggtttcttt tccctctagt aattctttta catttgtttt ggtgagtttt
     7141 ttctttctaa aatgttcagc taaagtaaac ttacattcag gataatttga acaaccataa
     7201 aacgattttt ttaatacaat attgttgcca cacttaggac attttcctac aatacttttt
     7261 tctgcttctt tttctttctg ttcctggtaa tcagaaaaat ttagtttttc tatatcgtta
     7321 ggtacagctt ccagtaaatg aacaatgaat tttttgatat tcgtaataaa gttctcttga
     7381 ttgccttctc ttttaccgat tttttttaaa tacgtttccc atttagccgt catttcagca
     7441 ctcgttaaaa ggtgctgact ttcaactgcc tggcacaata attttccttt ttcagttaca
     7501 acaagctcca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     7561 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     7621 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     7681 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct
     7741 tgcatgcctg caggtcgact ctagaggatc cccgggtacc gagctcgaat tcgcggccgc
     7801 gctagcaaag gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt
     7861 gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc tacatacgga
     7921 aagcttaccc ttaaatttat ttgcactact ggaaaactac ctgttccatg gccaacactt
     7981 gtcactactt tgacctatgg tgttcaatgc ttttcccgtt atccggatca tatgaaacgg
     8041 catgactttt tcaagagtgc catgcccgaa ggttatgtac aggaacgcac tatatctttc
     8101 aaagatgacg ggaactacaa gacgcgtgct gaagtcaagt ttgaaggtga tacccttgtt
     8161 aatcgtatcg agttaaaagg tattgatttt aaagaagatg gaaacattct cggacacaaa
     8221 ctcgagtaca actataactc acacaatgta tacatcacgg cagacaaaca aaagaatgga
     8281 atcaaagcta acttcaaaat tcgccacaac attgaagatg gatccgttca actagcagac
     8341 cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac
     8401 ctgtcgacac aatctgccct ttcgaaagat cccaacgaaa agcgtgacca catggtcctt
     8461 cttgagtttg taactgctgc tgggattaca catggcatgg atgagctcta caaataataa
     8521 ggccggccaa taaagatcct aggacgccgc caagccagct taaacccagc tcaatgagct
     8581 gggttttttg tttgttaaaa atgaagaaga aactgtgaag cgtatttaac tagtaaaatc
     8641 taagacatat catgatttat aaaaacaaaa aaaccttgca ggatatgcaa ggcttctgga
     8701 tgacccgtac gggattcgaa ctccggatgt gagcgccggt cgctaccatt accagttggt
     8761 ctggtgtcaa aaataataat aaccgggcag gccatgtctg cccgtatttc gcgtaaggaa
     8821 atccattatg tactatttaa ttctgcgtga catcccatcg atcagaccag tttttaattt
     8881 gtgtgtttcc atgtgtccag tttggaatac tcttaacctc attggaaatc gcggcataat
     8941 cactggtggt atgattgatg accgcgtcaa caatgacctt tatgccatat tcttcagcgg
     9001 ctgcacacat ttctttaaat tcttgttcag tacctaagta acggttgcca atttgatacg
     9061 atgtcggctg atacagccag taccagttcg acatgctttt atctccttga ttcccttcct
     9121 ttacttggtt aatcggagat gtctgaatgg ctgtatatcc tgcatcatga atatccttca
     9181 tattgtgttt taacgtattg aacgaccaat tccatgcatg aagaatggtt ccgcttttga
     9241 tcgacggtgc tgtaagctca ttcgatttgt tcgccgtttc agcactcgca gccgccggtc
     9301 ctgccagaac caaatgaaac agcaataaaa atccagcgaa taacggcagt aaagaggttt
     9361 tgaatcgttt tgcaaacatt cttgacactc cttatttgat tttttgaaga cttacttcgg
     9421 agtcaaaaat ccctcttact tcattcttcc gcttcctcct ttcaaaccga tgtgaagact
     9481 ggagaatttt gtt
//
