LOCUS       sequence-340973-        8391 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Plasmid contains the Cas12k gRNA and transposon donor cassette 
            assembled with eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 567 to 601)
  TITLE     Genetic manipulation in P. aeruginosa and Mycobacterium tuberculosis
REFERENCE   2  (bases 1 to 8391)
  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"
COMMENT     Sequence Label: pGTPA-eGFP
FEATURES             Location/Qualifiers
     source          1..8391
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..35
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     promoter        567..601
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     RBS             616..627
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(634..636,637..639,640..1353)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(679..700)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(940..959)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1287..1308
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    1357..1377
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      1378..1435
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     promoter        complement(1664..1682)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1665..1682)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1700..1716)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1700..1716)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1713..1735)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1724..1740
                     /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)."
     promoter        complement(join(1748..1754,1755..1772,1773..1778))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1793..1814
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1885..1903)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1971..2075
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2076..2144,2145..2936)
                     /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(2294..2313)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3274..3625
                     /label=pRO1600 oriV
                     /note="broad-host-range origin of replication from 
                     Pseudomonas aeruginosa plasmid pRO1600; requires the 
                     pRO1600 Rep protein for replication (West et al., 1994)"
     rep_origin      4929..5517
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5418..5437
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5671..5688
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5784..6239)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5921..5942)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6133..6152
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6365..6387
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6379..6396
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6380..6396
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6403..6422
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6403..6421
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     6451..6470
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(join(6509..7843,7844..7930))
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     regulatory      8367..8376
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 ttgacagcta gctcagtcct aggtataata ctagtatatt aatagcgccg caattcatgc
       61 tgcttgcagc ctctgaattt tgttaaatga gggttagttt gactgtataa atacagtctt
      121 gctttctgac cctggtagct gctcaccctg atgctgctgt caatagacag gataggtgcg
      181 ctcccagcaa taagggcgcg gatgtactgc tgtagtggct actgaatcac ccccgatcaa
      241 gggggaaccc tccaaaaggt gggttgaaag agaccattgg tctcattttt ttagccgtga
      301 tagtttgcga cagatatcga attcctgcag cccgggggat ccactagttg tacagtgact
      361 aattatatgt cgttgtgaca aattattgtc atcagtaaaa tccttataca gtatagatta
      421 tagcgctttg gcagttttag cataacctct ttgcagtgac aaaatagatg tcgttgtccg
      481 tgattgtgac aaattagctg tcgctttgca agataggaaa aagcttttgt gtattttcat
      541 aatgacaaat tgactgtcgc ctcgagttga cggctagctc agtcctaggt acagtgctag
      601 ctactagagc tagagaaaga ggagaaatac tagatggtga gcaagggcga ggagctgttc
      661 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
      721 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
      781 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
      841 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
      901 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
      961 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     1021 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     1081 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     1141 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     1201 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc caaactgagc
     1261 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     1321 atcactctcg gcatggacga gctgtacaag taataatact agtagcggcc gctgcagtcc
     1381 ggcaaaaaag ggcaaggtgt caccaccctg ccctttttct ttaaaaccga aaagatctag
     1441 atcagtaact attaaactta ggggtgggtt gaaagcaagt ccttttatcc gcttgtttta
     1501 attgctttgt ataataattg cagagcatat tatattgatg acatttaatt tgtcatcaat
     1561 taattaagca acgctgatgg gtcacgacga caattaaata gtcacaatga cattaatctg
     1621 tcaccgacga cagataattt gtcactgtac aaagcttgag tattctatag tgtcacctaa
     1681 atagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     1741 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     1801 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     1861 gccagaattc ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     1921 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     1981 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     2041 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     2101 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     2161 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     2221 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     2281 cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac
     2341 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     2401 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     2461 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     2521 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     2581 aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc
     2641 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     2701 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     2761 ttgctgataa atctggagcc ggtgagcgtg gatctcgcgg tatcattgca gcactggggc
     2821 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     2881 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     2941 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     3001 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     3061 cgttccactg agcgtcagac cccaattaca cgccactggc tgtgcttgct ggggtgacgg
     3121 tggcaacggt ggcggccttg ctgggctatc gcgttggaaa gaaacgaggg aaaggggact
     3181 gataaaccgg tcttagcccc tccccttggt gtccaaccgc tctgtaggcc tctcaggcgc
     3241 cgctggtgcc gctggttgga cgccaagggt gaatccgcct cgataccctg attactcgct
     3301 tcctgcgccc tctcaggcgg cgatagggga ctggtaaaac ggggattgcc cagacgcctc
     3361 ccccgcccct tcaggggcac aaatgcggcc ccaacggggc cacgtagtgg tgcgtttttt
     3421 gcgtttccac ccttttcttc cttttccctt ttaaaccttt taggacgtct acaggccacg
     3481 taatccgtgg cctgtagagt ttaaaaaggg acggatttgt tgccattaag ggacggattt
     3541 gttgttaaga agggacggat ttgttgttgt aaagggacgg atttgttgta ttgtgggacg
     3601 cagatacagt gtccccttat acacaaggaa tgtcgaacgt ggcctcaccc ccaatggttt
     3661 acaaaagcaa tgccctggtc gaggccgcgt atcgcctcag tgttcaggaa cagcggatcg
     3721 ttctggcctg tattagccag gtgaagagga gcgagcctgt caccgatgaa gtgatgtatt
     3781 cagtgacggc ggaggacata gcgacgatgg cgggtgtccc tatcgaatct tcctacaacc
     3841 agctcaaaga agcggccctg cgcctgaaac ggcgggaagt ccggttaacc caagagccca
     3901 atggcaaggg gaaaagaccg agtgtgatga ttaccggctg ggtgcaaaca atcatctacc
     3961 gggagggtga gggccgtgta gaactcaggt tcaccaaaga catgctgccg tacctgacgg
     4021 aactcaccaa acagttcacc aaatacgcct tggctgacgt ggccaagatg gacagcaccc
     4081 acgcgatcag gctttacgag ctgctcatgc aatgggacag catcggccag cgcgaaatag
     4141 aaattgacca gctgcgaaag tggtttcaac tggaaggccg gtatccctcg atcaaggact
     4201 tcaagttgcg agtgcttgat ccagccgtga cgcagatcaa cgagcacagc ccgctacagg
     4261 tggagtgggc gcagcgaaag accgggcgca aggtcacaca tctgttgttc agttttggac
     4321 cgaagaagcc cgccaaggcg gtgggtaagg ccccagcgaa gcgcaaggcc gggaagattt
     4381 cagatgctga gatcgcgaaa caggctcgcc ctggtgagac atgggaagcg gcccgcgctc
     4441 gactaaccca gatgccgctt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     4501 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     4561 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     4621 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     4681 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     4741 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     4801 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     4861 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     4921 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     4981 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     5041 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     5101 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     5161 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     5221 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     5281 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     5341 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     5401 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     5461 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     5521 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     5581 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     5641 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     5701 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg cacagatgcg
     5761 taaggagaaa ataccgcatc aggaaattgt aagcgttaat attttgttaa aattcgcgtt
     5821 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     5881 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     5941 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     6001 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     6061 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     6121 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     6181 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     6241 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     6301 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
     6361 ttttcccagt cacgacgttg taaaacgacg gccagtgaat tgtaatacga ctcactatag
     6421 ggcgaattcg agctcggtac ccggggatcc cggcatcaga gcagattgta ctgagagtgc
     6481 accataatcg gcattttctt ttgcgttttt atttgttaac tgttaattgt ccttgttcaa
     6541 ggatgctgtc tttgacaaca gatgttttct tgcctttgat gttcagcagg aagctaggcg
     6601 caaacgttga ttgtttgtct gcgtagaatc ctctgtttgt catatagctt gtaatcacga
     6661 cattgtttcc tttcgcttga ggtacagcga agtgtgagta agtaaaggtt acatcgttag
     6721 gatcaagatc catttttaac acaaggccag ttttgttcag cggcttgtat gggccagtta
     6781 aagaattaga aacataacca agcatgtaaa tatcgttaga cgtaatgccg tcaatcgtca
     6841 tttttgatcc gcgggagtca gtgaacagat accatttgcc gttcatttta aagacgttcg
     6901 cgcgttcaat ttcatctgtt actgtgttag atgcaatcag cggtttcatc acttttttca
     6961 gtgtgtaatc atcgtttagc tcaatcatac cgagagcgcc gtttgctaac tcagccgtgc
     7021 gttttttatc gctttgcaga agtttttgac tttcttgacg gaagaatgat gtgcttttgc
     7081 catagtatgc tttgttaaat aaagattctt cgccttggta gccatcttca gttccagtgt
     7141 ttgcttcaaa tactaagtat ttgtggcctt tatcttctac gtagtgagga tctctcagcg
     7201 tatggttgtc gcctgagctg tagttgcctt catcgatgaa ctgctgtaca ttttgatacg
     7261 tttttccgtc accgtcaaag attgatttat aatcctctac accgttgatg ttcaaagagc
     7321 tgtctgatgc tgatacgtta acttgtgcag ttgtcagtgt ttgtttgccg taatgtttac
     7381 cggagaaatc agtgtagaat aaacggattt ttccgtcaga tgtaaatgtg gctgaacctg
     7441 accattcttg tgtttggtct tttaggatag aatcatttgc atcgaatttg tcgctgtctt
     7501 taaagacgcg gccagcgttt ttccagctgt caatagaagt ttcgccgact ttttgataga
     7561 acatgtaaat cgatgtgtca tccgcatttt taggatctcc ggctaatgca aagacgatgt
     7621 ggtagccgtg atagtttgcg acagtgccgt cagcgttttg taatggccag ctgtcccaaa
     7681 cgtccaggcc ttttgcagaa gagatatttt taattgtgga cgaatcgaac tcaggaactt
     7741 gatatttttc atttttttgc tgttcaggga tttgcagcat atcatggcgt gtaatatggg
     7801 aaatgccgta tgtttcctta tatggctttt ggttcgtttc tttcgcaaac gcttgagttg
     7861 cgcctcctgc cagcagtgcg gtagtaaagg ttaatactgt tgcttgtttt gcaaactttt
     7921 tgatgttcat cgttcatgtc tcctttttta tgtactgtgt tagcggtctg cttcttccag
     7981 ccctcctgtt tgaagatggc aagttagtta cgcacaataa aaaaagacct aaaatatgta
     8041 aggggtgacg ccaaagtata cactttgccc tttacacatt ttaggtcttg cctgctttat
     8101 cagtaacaaa cccgcgcgat ttacttttcg acctcattct attagactct cgtttggatt
     8161 gcaactggtc tattttcctc ttttgtttga tagaaaatca taaaaggatt tgcagactac
     8221 gggcctaaag aactaaaaaa tctatctgtt tcttttcatt ctctgtattt tttatagttt
     8281 ctgttgcatg ggcataaagt tgcgtcgacg ccttgctcaa attggaatca ggtttgtgcc
     8341 aataccagta gaaacagacg aagaatccat ggcggccgct gtcatgggcc c
//
