LOCUS       sequence-333401-        6202 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Suicide vector for integrating expression cassette in P. putida 
            (IPTG induction).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6202)
  AUTHORS   Cook TB, Jacobson TB, Venkataraman MV, Hofstetter H, Amador-Noguez 
            D, Thomas MG, Pfleger BF
  TITLE     Stepwise genetic engineering of Pseudomonas putida enables robust 
            heterologous production of prodigiosin and glidobactin A.
  JOURNAL   Metab Eng. 2021 Jun 24;67:112-124. doi: 10.1016/j.ymben.2021.06.004.
  PUBMED    34175462
REFERENCE   2  (bases 1 to 6202)
  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: "Metab 
            Eng."; date: "2021-06-24"; volume: "67:112-124. doi"; pages: " 
            10.1016/j.ymben.2021.06.004"
COMMENT     Sequence Label: pNVLTv2-pvdIJD-Ptrc-base
FEATURES             Location/Qualifiers
     source          1..6202
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(64..81)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(198..743)
                     /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(278..297)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     819..838
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     oriT            966..1074
                     /note="incP origin of transfer"
     primer_bind     1104..1122
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             1429..2223
                     /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(1483..1502)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2093..2112
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     promoter        3669..3703
                     /label=BBa_J23101
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.7"
     CDS             3730..4812
                     /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     complement(3749..3768)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5044..5049,5050..5066,5067..5073)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    5081..5097
                     /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)."
ORIGIN
        1 catagctgtt tcctgtgtga aattgcgtca gcagaatatg tgatacagga tatattccgc
       61 ttcctcgctc actgactcgc tacgctcggt cgttcgactg cggcgagcgg aaatggctta
      121 cgaacggggc ggagatttcc tggaagatgc caggaagata cttaacaggg aagtgagagg
      181 gccgcggcaa agccgttttt ccataggctc cgcccccctg acaagcatca cgaaatctga
      241 cgctcaaatc agtggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
      301 ggcggctccc tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt cattccgctg
      361 ttatggccgc gtttgtctca ttccacgcct gacactcagt tccgggtagg cagttcgctc
      421 caagctggac tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct tatccggtaa
      481 ctatcgtctt gagtccaacc cggaaagaca tgcaaaagca ccactggcag cagccactgg
      541 taattgattt agaggagtta gtcttgaagt catgcgccgg ttaaggctaa actgaaagga
      601 caagttttgg tgactgcgct cctccaagcc agttacctcg gttcaaagag ttggtagctc
      661 agagaacctt cgaaaaaccg ccctgcaagg cggttttttc gttttcagag caagagatta
      721 cgcgcagacc aaaacgatct caagaagatc atcttattaa tcagataaaa tatttctagg
      781 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
      841 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc
      901 tgctcatgtt tgacagctta tcgctcagtg gaacgaaaac tcgcatgtat ccaacggcgt
      961 cagccgggca ggataggtga agtaggccca cccgcgagcg ggtgttcctt cttcactgtc
     1021 ccttattcgc acctggcggt gctcaacggg aatcctgctc tgcgaggctg gccgtagtgc
     1081 ttggattctc accaatatat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt
     1141 tcggtgatga cggtgaaaac ctctgagatg aatgtcagct actgggctat ctggacaagg
     1201 gaaaacgcaa gcgcaaagag aaagcaggta gcttgcagtg ggcttacatg gcgatagcta
     1261 gactgggcgg ttttatggac agcaagcgaa ccggaattgc cagctggggc gccctctggt
     1321 aaggttggga agccctgcaa agtaaactgg atggctttct tgccgccaag gatctgatgg
     1381 cgcaggggat caagatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
     1441 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     1501 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     1561 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
     1621 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     1681 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     1741 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     1801 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     1861 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     1921 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc
     1981 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     2041 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     2101 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     2161 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     2221 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg tcatcacgag
     2281 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     2341 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccgggc
     2401 tcgatcccct cgcgagttgg ttcagctgct gcctgaggct ggacgacctc gcggagttct
     2461 accggcagtg caaatccgtc ggcatccagg aaaccagcag cggctatccg cgcatccatg
     2521 cccccgaact gcaggagtgg ggaggcacga tggccgcttt ggtcccggat cctctagagt
     2581 cgacctgcag gcatgcaagc ttggcgtaat cagctggacg tctttgacga actcaaggct
     2641 atcgtcgatg gccacctcga ccagtgcggc ggggtactgc tgcgtggttt cgacgtgggc
     2701 ggagtcacgg cctttcgcga attcgcccag gccttcggcc acccgctgct caactacgag
     2761 ttcggctcga cgccgcgcag caatgttacc caaggggtct acacctccac ggagtacccc
     2821 gcgcaccaga gcatcccgct gcacaacgag caggcctaca gcctcgactg gccgatgcgg
     2881 atttggttct acagtgtgat cgccgctcag actggcggtg aaacgccaat tgccgacagc
     2941 cgtgaagtgt tccggcgcat gccggccgac atacgcgagc gctttgccag caagcgcctg
     3001 atgtatgtgc gcaactacgg caatggcctt gatgtgagtt gggagcaggt gttcaacacc
     3061 gaggaccgca gcgtagtgga ggcctattgc cgcgcgcacc acatcgactg ggaatggaag
     3121 gacgacggtg agctgcgcac gcgccaggtc tgccaggccg tggccacgca cccctggacg
     3181 ggggatcagg tatggttcaa tcaggcccac ctgttccatg tctccaacct accggccgag
     3241 gtgcgcgaaa gcctgctgga aatcgtcgac gaggaagact tgccgcgcaa cgtctactac
     3301 ggcgacggca caccgatcga gaacgaagtg ctcgaacgca ttcgcgctgt gctggacgat
     3361 tgcgcgatca gtttcccctg gcaggaaaac gacgtgctga tgctcgacaa catgctcgcg
     3421 gcgcacgccc gggcaccgtt caccgggcca cgcaaagtgg tcgtggccat ggctcagggg
     3481 caccgcgaag cggcgctttg aggccactgt ggcgagtgct tgcggcttgc cgcaggcgct
     3541 cgccagcgca gcgtccgggc atggcgctgc cgcgttaaat tcgccctccc tccgctcgtt
     3601 ctactggaaa cgctttggcc gcacggccga gctgcagatc agtaaggacc ccgagcgaca
     3661 ccatcgaatt tacagctagc tcagtcctag gtattatgct agcaagagag tcaattcagg
     3721 gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg tgtctcttat
     3781 cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa
     3841 gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca acaactggcg
     3901 ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca cgcgccgtcg
     3961 caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt agtggtgtcg
     4021 atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa
     4081 cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat tgctgtggaa
     4141 gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac
     4201 agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg
     4261 ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt
     4321 ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa
     4381 ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc
     4441 gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt
     4501 accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa
     4561 gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg
     4621 caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag
     4681 ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc
     4741 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     4801 agcgggcagt gagcgcaacg caattaatgt aagttagcgc gaattgatct ggtttgacag
     4861 cttatcatcg actgcacggt gcaccaatgc ttctggcgtc aggcagccat cggaagctgt
     4921 ggtatggctg tgcaggtcgt aaatcactgc ataattcgtg tcgctcaagg cgcactcccg
     4981 ttctggataa tgttttttgc gccgacatca taacggttct ggcaaatatt ctgaaatgag
     5041 ctgttgacaa ttaatcatcc ggctcgtata atgtgtggaa ttgtgagcgg ataacaattt
     5101 caggtaccct cgagtaagga tctccagacg acgtacaagc gctgctcgaa cccaacctca
     5161 cgcacgcctg cttcattgaa ctttcatgag ctttcggggc cgccagcgcg gcccctctgg
     5221 agaatcgaac gatgtccctg aaccctgacc tggcggccta cctgcaactg gtggaggccg
     5281 gacgcagcgc cggcaaggtg ctgcccatgc atgccctggc ggcggacgag gcgcgccgcc
     5341 agttcgaaga gtcctcggcg ctgattgcgg gcaaggccga cgagccggac tgcatcagcg
     5401 acctttcgtt gaccacccgc gatggccata ccttgccggt gcgcttgtac cgcccgccgc
     5461 aggccgaccc tgcactggcc ggcgcggcgc tgctgtacct gcatggcggc ggctacgtgg
     5521 tgggcagcct cgattcgcat gacacgctgt gctggaacct ggcgcaggat gccggggtgc
     5581 cggtgattgc cgtgggctac cgactggcgc cgcagtggcg tttcccgaca gccagtgacg
     5641 atgccctgga tgcctggcgc tggttggtcg agcaggccga ggcgctgggc atcgatgcgc
     5701 agcgactggc ggtggtgggc gacagtgtcg gtggtagcct ggctaccatt ctggccaacc
     5761 agttggctgc gcagcgcgag ctgccggcac cgcgcctgca ggtaatgatc tacccggtga
     5821 ccgatgccag ttgccggcgg ccttcggtgc agcgctatgg cagcggttac ctgcttgaag
     5881 cgcagaccct ggagtggttc taccagcaat acgccacggt gccggcagac cgtcacgatc
     5941 cgcgtttttc accgttgctg ggcagcgtcg ccagcaacag tgcacctgca ctgatgctga
     6001 ttgccgagtg cgacccgctg catgaccagg gtgtcgctta tgcacggcac ttggagcaag
     6061 ccggggtggc ggtgcagctt gcggtgatcc cgggagtgac ccatgacttc atgcgtatgg
     6121 gttcgatcat cgaggaggcc gatgaaggcc tggcgatggt ggtcgaggca ctgcagcagc
     6181 atctgtaagg gagcctttca gg
//
