LOCUS       sequence-439084-        6288 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector with GFP expressed under PabstBR promoter, kanR 
            marker, and replicates in A. baumannii and E. coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6288)
  AUTHORS   Bacon EE, Tran JS, Nadig N, Peters JM
  TITLE     Modular, inducible, and titratable expression systems for 
            Escherichia coli and Acinetobacter baumannii.
  JOURNAL   Microbiol Spectr. 2024 Nov 5;12(11):e0130624. doi: 
            10.1128/spectrum.01306-24. Epub 2024 Sep 20.
  PUBMED    39302127
REFERENCE   2  (bases 1 to 6288)
  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: "Microbiol 
            Spectr."; date: "2024-11-5"; volume: "12(11):e0130624. doi"; pages: 
            " 10.1128/spectrum.01306-24. Epub 2024 Sep 20"
COMMENT     Sequence Label: pJMP3654
FEATURES             Location/Qualifiers
     source          1..6288
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     oriT            588..697
                     /note="incP origin of transfer"
     CDS             complement(2320..3114)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2431..2450)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3041..3060
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(3225..3230,3231..3247,3248..3253))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     terminator      complement(3329..3356)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3448..3534)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3687..3704
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(join(3772..4482,4483..4485))
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             complement(3772..4485)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(3812..3832)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     4427..4455
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     CDS             complement(4702..5784)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5746..5765
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5785..5862)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     rep_origin      complement(join(5948..6288,1..205))
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(6028..6047)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggtaattgat ttagaggagt tagtcttgaa gtcatgcgcc ggttaaggct aaactgaaag
       61 gacaagtttt ggtgactgcg ctcctccaag ccagttacct cggttcaaag agttggtagc
      121 tcagagaacc ttcgaaaaac cgccctgcaa ggcggttttt tcgttttcag agcaagagat
      181 tacgcgcaga ccaaaacgat ctcaagaaga tcatcttatt aatcagataa aatatttcta
      241 gctagagtcg atcttcgcca gcagggcgag gatcgtggca tcaccgaacc gcgccgtgcg
      301 cgggtcgtcg gtgagccaga gtttcagcag gccgcccagg cggcccaggt cgccattgat
      361 gcgggccagc tcgcggacgt gctcatagtc cacgacgccc gtgattttgt agccctggcc
      421 gacggccagc aggtaggccg acaggctcat gccggccgcc gccgcctttt cctcaatcgc
      481 tcttcgttcg tctggaaggc agtacacctt gataggtggg ctgcccttcc tggttggctt
      541 ggtttcatca gccatccgct tgccctcatc tgttacgccg gcggtagccg gccagcctcg
      601 cagagcagga ttcccgttga gcaccgccag gtgcgaataa gggacagtga agaaggaaca
      661 cccgctcgcg ggtgggccta cttcacctat cctgcccggc tgacgccgtt ggatacacca
      721 aggaaagtct acacgaaccc tttggcaaaa tcctgtatat cgtgcgaaaa aggatggata
      781 taccgaaaaa atcgctataa tgaccccgaa gcagggttat gcagcggaaa agcgctgctt
      841 ccctgctgtt ttgtgcgcgc ctgaggacat aagggctgca agccaagatc gtagaaatat
      901 ctatgattat cttgaagaac gcaaccctat agcagctatt gaaattgatg atttaattga
      961 agaaaagaca gatttacttg ttgataatcg actgatgggg cgcacaggca gacagaaaga
     1021 tactagggag ttagtgatac atccgcatta tgtggttgta tatgacatca ctgatataat
     1081 acggatactc agagtgctac acacatcgca ggagtggtca tgacttactc atgtactttg
     1141 gattatttag tgttataaaa tcctgattta taaatttttt ttgttaaaaa agataaaagc
     1201 cccttgcaat tgcttggggc tttaccgtaa tttatggggt acagatcttc gatactgaca
     1261 tatcggcaat cgaaagcatt aaggtttgac gaccgctaat gatttcacca caggggctta
     1321 atgtacctgt cttaaattct aaggttttaa ctcgctttgt caagcataga ccccaaaaat
     1381 ttagccaatg tctgtaactc aatctgtcca tgtgtgggtg atgaggtaca gtgacgctag
     1441 cacacatcgg aaaaacgcta ttactagggg aactgaacag agtagcggac gcaatgagta
     1501 gtcatttaat tggcggttat gagcgtgttc aggcggtgct atcaatcgta atcataacag
     1561 tggcagcttg atacagtgat gtcatccctg atgcgaaagc gaccgaccga cggtacatcg
     1621 aatgggaata ctttagggtg atttttaaga atcgctctag ggtgagtatt tcccattcag
     1681 ctctgctctc tccctctggt actttaatca aaagcactac taaacatatg tttttaaata
     1741 aaaaatattg atatagagat aatattagta agaataatta aacaattgaa tatagataaa
     1801 tcattgttaa ataaagatta attattaaaa tgaatgcata cttatatata aatcaatgat
     1861 ttaaaatatt tgataaagaa aacttttcaa aaaaaatata attgagattg tgtcatttcg
     1921 gtcaattctt aatatgttcc acgcaagttt tagctatggt gctaaacaga aatttgctga
     1981 aaaagaactt ttcactgaac tggttaaaat gtaagcagcc tgagagccgc caaaaatttt
     2041 aaaaacaaac cgccttaatc atcttcaaaa aatacctcta aaacctcacc atttgcgttt
     2101 taagacccat atttcatcct gcccttatgt tcccatgctg atagctataa agtgtctgta
     2161 atcgcttcct atgacgttct aggctgttga taacttttgg aacaacgcaa aatgttaaaa
     2221 tcctgctgtg ataatgtcaa cggactagtc cagaaaccgc agtacatcgg agcgcttttg
     2281 aagctgatgc tcgagtcatt tcgaacccca gagtcccgct cagaagaact cgtcaagaag
     2341 gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg
     2401 gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg ctatgtcctg
     2461 atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc ggccattttc
     2521 caccatgata ttcggcaagc aggcatcgcc gtgggtcacg acgagatcct cgccgtcggg
     2581 catgcgcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat gctcttcgtc
     2641 cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct cgatgcgatg
     2701 tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc
     2761 atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga gatcctgccc
     2821 cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt cgagcacagc
     2881 tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt cctgcagttc
     2941 attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct gcgctgacag
     3001 ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat agccgaatag
     3061 cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa tcatgcgaaa
     3121 cgatcctcat cctgtctctt gatcagatct tgatcccctg cgccatcaga tccttggcgg
     3181 caagaaagcc atccagtttt gagcgcatac gctacttgca ttacagttta cgaaccgaac
     3241 aggcttatgt caactgggtt cgtgccttca tccgtttcca cggctcgaga gttcaacaga
     3301 cagccttcgt tcttatggcc gttcaggaaa aaggccatcc gtcaggatgg ccttctgctt
     3361 aatttgatgc ctggcagttt atggcgggcg tcctgcccgc caccctccgg gccgttgctt
     3421 cgcaacgttc aaatccgctc ccggcggatt tgtcctactc aggagagcgt tcaccgacaa
     3481 acaacagata aaacgaaagg cccagtcttt cgactgagcc tttcgtttta tttgatgcct
     3541 ggcagttccc tactctcgca tggggagacc ccacactacc atcggcgcta cggcgtttca
     3601 cttctgagtt cggcatgggg tcaggtggga ccaccgcgct actgccgcca ggcaaattct
     3661 gttttatcag accgcttctg cgttctgatt taatctgtat caggctgaaa atcttctctc
     3721 atccgccaaa acagccaagc ttgcatgcct gcaggtcgac tctagaggat ctttgtagag
     3781 ctcatccatg ccatgtgtaa tcccagcagc agttacaaac tcaagaagga ccatgtggtc
     3841 acgcttttcg ttgggatctt tcgaaaggac agattgtgtc gacaggtaat ggttgtctgg
     3901 taaaaggaca gggccatcgc caattggagt attttgttga taatggtctg ctagttgaac
     3961 ggaaccatct tcaacgttgt ggcgaatttt gaagttagct ttgattccat tcttttgttt
     4021 gtctgccgtg atatatacat tgtgtgagtt aaagttgtac tccagtttgt gtccaagaat
     4081 gtttccatct tctttaaaat caataccctt taactcgata cgattaacaa gggtatcacc
     4141 ttcaaacttg acttcagcac gcgtcttgta ggtcccgtca tctttgaaag atatagtgcg
     4201 ttcctgtaca taaccttcgg gcatggcact cttgaaaaag tcatgccgtt tcatgtgatc
     4261 cggataacgg gaaaagcatt gaacaccata ggtcagagta gtgacaagtg ttggccacgg
     4321 aacaggtagt tttccagtag tgcaaataaa tttaagggtg agttttccgt ttgtagcatc
     4381 accttcaccc tctccacgga cagaaaattt gtgcccatta acatcaccat ctaattcaac
     4441 aagaattggg acaactccag tgaaaagttc ttctcctttg ctcatggtct gtttcctgtg
     4501 tgctgctgat gtgctcagta tcttgttatc cgctcacatt gtcaatattt tatctgatta
     4561 atgagacgtc tttacgacct gcacagccat accacagctt ccgatggctg cctgacgcca
     4621 gaagcattgg tgcaccgtgc agtcgatgat aagctgtcaa accagatcaa ttcgcgctaa
     4681 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4741 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     4801 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
     4861 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
     4921 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
     4981 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
     5041 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
     5101 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
     5161 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
     5221 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
     5281 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
     5341 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
     5401 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
     5461 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
     5521 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
     5581 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
     5641 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
     5701 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
     5761 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     5821 tcatgccata ccgcgaaagg ttttgcacca ttcgatggtg tcaacgtaaa tgcatgccgc
     5881 ttcgccttcc gtttgggcct agaattccgg tttaacaggg aagtgagagg gccgcggcaa
     5941 agccgttttt ccataggctc cgcccccctg acaagcatca cgaaatctga cgctcaaatc
     6001 agtggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggcggctccc
     6061 tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt cattccgctg ttatggccgc
     6121 gtttgtctca ttccacgcct gacactcagt tccgggtagg cagttcgctc caagctggac
     6181 tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct tatccggtaa ctatcgtctt
     6241 gagtccaacc cggaaagaca tgcaaaagca ccactggcag cagccact
//
