LOCUS       sequence-344754-        6078 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of J1-BBa_J23117-mRFP and hAAVS1 scRNA on pRK2-GmR 
            plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6078)
  AUTHORS   Kiattisewee C, Dong C, Fontana J, Sugianto W, Peralta-Yahya P, 
            Carothers JM, Zalatan JG
  TITLE     Portable bacterial CRISPR transcriptional activation enables 
            metabolic engineering in Pseudomonas putida.
  JOURNAL   Metab Eng. 2021 Apr 27. pii: S1096-7176(21)00063-X. doi: 
            10.1016/j.ymben.2021.04.002.
  PUBMED    33930546
REFERENCE   2  (bases 1 to 6078)
  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. 
            2021 Apr 27. pii: S1096-7176(21)00063-X. doi: 
            10.1016/j.ymben.2021.04.002."
COMMENT     Sequence Label: pPPC018.AAV
FEATURES             Location/Qualifiers
     source          1..6078
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(join(314..319,320..336,337..342))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     primer_bind     848..870
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     862..879
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     863..879
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     889..908
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        889..907
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        940..974
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        1058..1076
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     CDS             1099..1128
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1144..1161
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1217..1234)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1387..1433
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1478..1498)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1478..1496)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1517..1533)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1517..1533)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1530..1552)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1541..1557
                     /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(1565..1571,1572..1589,1590..1595))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1610..1631
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        1924..1958
                     /label=BBa_J23117
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.06"
     RBS             1968..1979
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1986..2663
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      2690..2761
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2777..2804
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(3121..4269)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     IVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     rep_origin      4662..5193
                     /label=oriV
                     /note="incP origin of replication"
     terminator      complement(5305..5399)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(5670..6078,1..125))
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEVVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
ORIGIN
        1 gcccgcatgg atttgacttg gtcagggccg agcctacatg tgcgaatgat gcccatactt
       61 gagccaccta actttgtttt agggcgactg ccctgctgcg taacatcgtt gctgctgcgt
      121 aacatcgttg ctgctccata acatcaaaca tcgacccacg gcgtaacgcg cttgctgctt
      181 ggatgcccga ggcatagact gtacaaaaaa acagtcataa caagccatga aaaccgccac
      241 tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac cagttgcgtg agcgcatacg
      301 ctacttgcat tacagtttac gaaccgaaca ggcttatgtc aactgggttc gtgccttcat
      361 ccgtttccac ggtgtgcgtc catgggcaaa tattatacgc aaggcgacaa ggtgctgatg
      421 ccgctggcga ttcaggttca tcatgccgtt tgtgatggct tccatggctt cccaacctta
      481 ccagagggcg ccccagctgg caattccgac gtcgtcgaat ttgctttcga atttctgcca
      541 ttcatccgct tattatcact tattcaggcg tagcaaccag gcgtttaagg gcaccaataa
      601 ctgccttaaa aaaattacgc cccgccctgc cactcatcgc agtacggcct attggttaaa
      661 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
      721 ttccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
      781 ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca
      841 gggttttccc agtcacgacg ttgtaaaacg acggccagtg agcgcgcgta atacgactca
      901 ctatagggcg aattggagct ccaccgcggt ggcggccgct tgacagctag ctcagtccta
      961 ggtataatac tagtggggcc actagggaca ggatgtttta gagctagaaa tagcaagtta
     1021 aaataaggct agtccgttat caacttgaaa aagtggcaca tgaggatcac ccatgtgctt
     1081 tttttgaagc ttgggcccga acaaaaactc atctcagaag aggatctgaa tagcgccgtc
     1141 gaccatcatc atcatcatca ttgagtttaa acggtctcca gcttggctgt tttggcggat
     1201 gagagaagat tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac
     1261 agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag
     1321 tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc
     1381 aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt
     1441 ttgtcggtga actgggcccg gtacccagct tttgttccct ttagtgaggg ttaattgcgc
     1501 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     1561 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     1621 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     1681 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgcatgg
     1741 caattccgac gtcgcctacg gtatccaccg gagacctatg gcagcctccg gccgccatag
     1801 gacacctttg gttgccaagg gtgacctatg gtgaccatgg gccaccacgg gcgacctcag
     1861 gtatcctgcg gtgtcctgcg gttaccaaag gcgtcctttg ggttccaccg gatacctccg
     1921 gacttgacag ctagctcagt cctagggatt gtgctagcga attcattaaa gaggagaaag
     1981 gtaccatggc gagtagcgaa gacgttatca aagagttcat gcgtttcaaa gttcgtatgg
     2041 aaggttccgt taacggtcac gagttcgaaa tcgaaggtga aggtgaaggt cgtccgtacg
     2101 aaggtaccca gaccgctaaa ctgaaagtta ccaaaggtgg tccgctgccg ttcgcttggg
     2161 acatcctgtc cccgcagttc cagtacggtt ccaaagctta cgttaaacac ccggctgaca
     2221 tcccggacta cctgaaactg tccttcccgg aaggtttcaa atgggaacgt gttatgaact
     2281 tcgaagacgg tggtgttgtt accgttaccc aggactcctc cctgcaagac ggtgagttca
     2341 tctacaaagt taaactgcgt ggtaccaact tcccgtccga cggtccggtt atgcagaaaa
     2401 aaaccatggg ttgggaagct tccaccgaac gtatgtaccc ggaagacggt gctctgaaag
     2461 gtgaaatcaa aatgcgtctg aaactgaaag acggtggtca ctacgacgct gaagttaaaa
     2521 ccacctacat ggctaaaaaa ccggttcagc tgccgggtgc ttacaaaacc gacatcaaac
     2581 tggacatcac ctcccacaac gaagactaca ccatcgttga acagtacgaa cgtgctgaag
     2641 gtcgtcactc caccggtgct taaaaactcg agtaaggatc tccaggcatc aaataaaacg
     2701 aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct
     2761 ctactagagt cacactggct caccttcggg tgggcctttc tgcgtttata taaaaactgt
     2821 tgtaattcat taagcattct gccgacatgg aagccatcac aaacggcatg atgaacctga
     2881 atcgccagcg gcatcagcac cttgtcgcct tgcgtataat atttgcgaat tcttcagcca
     2941 aaaaacttaa gaccgccggt cttgtccact accttgcagt aatgcggtgg acaggatcgg
     3001 cggttttctt ttctcttctc aacctaggct acagccgata gtctggaaca gcgcacttac
     3061 gggacctagg gcgatgcagg tggctgctga acccccagcc ggaactgacc ccacaaggcc
     3121 ctagcgtttg caatgcacca ggtcatcatt gacccaggcg tgttccacca ggccgctgcc
     3181 tcgcaactct tcgcaggctt cgccgacctg ctcgcgccac ttcttcacgc gggtggaatc
     3241 cgatccgcac atgaggcgga aggtttccag cttgagcggg tacggctccc ggtgcgagct
     3301 gaaatagtcg aacatccgtc gggccgtcgg cgacagcttg cggtacttct cccatatgaa
     3361 tttcgtgtag tggtcgccag caaacagcac gacgatttcc tcgtcgatca ggacctggca
     3421 acgggacgtt ttcttgccac ggtccaggac gcggaagcgg tgcagcagcg acaccgattc
     3481 caggtgccca acgcggtcgg acgtgaagcc catcgccgtc gcctgtaggc gcgacaggca
     3541 ttcctcggcc ttcgtgtaat accggccatt gatcgaccag cccaggtcct ggcaaagctc
     3601 gtagaacgtg aaggtgatcg gctcgccgat aggggtgcgc ttcgcgtact ccaacacctg
     3661 ctgccacacc agttcgtcat cgtcggcccg cagctcgacg ccggtgtagg tgatcttcac
     3721 gtccttgttg acgtggaaaa tgaccttgtt ttgcagcgcc tcgcgcggga ttttcttgtt
     3781 gcgcgtggtg aacagggcag agcgggccgt gtcgtttggc atcgctcgca tcgtgtccgg
     3841 ccacggcgca atatcgaaca aggaaagctg catttccttg atctgctgct tcgtgtgttt
     3901 cagcaacgcg gcctgcttgg cttcgctgac ctgttttgcc aggtcctcgc cggcggtttt
     3961 tcgcttcttg gtcgtcatag ttcctcgcgt gtcgatggtc atcgacttcg ccaaacctgc
     4021 cgcctcctgt tcgagacgac gcgaacgctc cacggcggcc gatggcgcgg gcagggcagg
     4081 gggagccagt tgcacgctgt cgcgctcgat cttggccgta gcttgctgga ctatcgagcc
     4141 gacggactgg aaggtttcgc ggggcgcacg catgacggtg cggcttgcga tggtttcggc
     4201 atcctcggcg gaaaaccccg cgtcgatcag ttcttgcctg tatgccttcc ggtcaaacgt
     4261 ccgattcatt caccctcctt gcgggattgc cccggaatta attccccgga tcgatccgtc
     4321 gatcttgatc ccctgcgcca tcagatcctt ggcggcaaga aagccatcca gtttactttg
     4381 cagggcttcc caaccttacc agagggcgcc ccagctggca attccggttc gcttgctgtc
     4441 cataaaaccg cccagtctag ctatcgccat gtaagcccac tgcaagctac ctgctttctc
     4501 tttgcgcttg cgttttccct tgtccagata gcccagtagc tgacattcat ccggggtcag
     4561 caccgtttct gcggactggc tttctacgtg gctgccattt ttggggtgag gccgttcgcg
     4621 gccgaggggc gcagcccctg gggggatggg aggcccgcgt tagcgggccg ggagggttcg
     4681 agaagggggg gcacccccct tcggcgtgcg cggtcacgcg cacagggcgc agccctggtt
     4741 aaaaacaagg tttataaata ttggtttaaa agcaggttaa aagacaggtt agcggtggcc
     4801 gaaaaacggg cggaaaccct tgcaaatgct ggattttctg cctgtggaca gcccctcaaa
     4861 tgtcaatagg tgcgcccctc atctgtcagc actctgcccc tcaagtgtca aggatcgcgc
     4921 ccctcatctg tcagtagtcg cgcccctcaa gtgtcaatac cgcagggcac ttatccccag
     4981 gcttgtccac atcatctgtg ggaaactcgc gtaaaatcag gcgttttcgc cgatttgcga
     5041 ggctggccag ctccacgtcg ccggccgaaa tcgagcctgc ccctcatctg tcaacgccgc
     5101 gccgggtgag tcggcccctc aagtgtcaac gtccgcccct catctgtcag tgagggccaa
     5161 gttttccgcg aggtatccac aacgccggcg gccctacatg gctctgctgt agtgagtggg
     5221 ttgcgctccg gcagcggtcc tgatcccccg cagaaaaaaa ggatctcaag aagatccttt
     5281 gatcttttct acactagtgc ttggattctc accaataaaa aacgcccggc ggcaaccgag
     5341 cgttctgaac aaatccagat ggagttctga ggtcattact ggatctatca acaggagtcc
     5401 aagcgagctc accttcggga gcgcctgaag cccgttctgg acgccctggg gccgttgaat
     5461 cgggatatgc aggccaaggc cgccgcgatc atcaaggccg tgggcgaaaa gctgctgacg
     5521 gaacagcggg aagtccagcg ccagaaacag gcccagcgcc agcaggaacg cgggcgcgca
     5581 catttccccg aaaagtgcca cctgcggcgt tgtgacaatt taccgaacaa ctccgcggcc
     5641 gggaagccga tctcggcttg aacgaattgt taggtggcgg tacttgggtc gatatcaaag
     5701 tgcatcactt cttcccgtat gcccaacttt gtatagagag ccactgcggg atcgtcaccg
     5761 taatctgctt gcacgtagat cacataagca ccaagcgcgt tggcctcatg cttgaggaga
     5821 ttgatgagcg cggtggcaat gccctgcctc cggtgctctc cggagactgc gagatcatag
     5881 atatagatct cactacgcgg ctgctcaaac ttgggcagaa cgtaagccgc gagagcgcca
     5941 acaaccactt cttggtcgaa ggcagcaagc gcgatgaatg tcttactacg gagcaagttc
     6001 ccgaggtaat cggagtccgg ctgatgttgg gagtaggtgg ctacgtctcc gaactcacga
     6061 ccgaaaagat caagagca
//
