LOCUS       sequence-110843-        6276 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  encodes a reversed Int7 gene flanked by the attB/P sites of Int5, as
            well as a reversed GFP (GFPmut33) gene flanked by the attB/P sites 
            of Int7. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6276)
  AUTHORS   Yang L, Nielsen AA, Fernandez-Rodriguez J, McClune CJ, Laub MT, Lu 
            TK, Voigt CA
  TITLE     Permanent genetic memory with >1-byte capacity.
  JOURNAL   Nat Methods. 2014 Dec;11(12):1261-6. doi: 10.1038/nmeth.3147. Epub 
            2014 Oct 26.
  PUBMED    25344638
REFERENCE   2  (bases 1 to 6276)
  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: 
            "10.1038/nmeth.3147"; journalName: "Nat Methods"; date: "2014-12"; 
            volume: "11"; issue: "12"; pages: "1261-6"
COMMENT     Sequence Label: pCas_5+7_gfp
FEATURES             Location/Qualifiers
     source          1..6276
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        28..62
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     CDS             complement(182..895)
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MSKGEELFTGVVPILVELDGNVNGHKFSVSGEGEGDAAYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(222..242)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     837..865
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     RBS             902..913
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     misc_RNA        complement(944..993)
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     terminator      complement(1106..1192)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(1561..2105)
                     /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(1641..1660)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(2190..2284)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(2305..2964)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2879..2898
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      complement(3127..3156)
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     primer_bind     3220..3238
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(3242..3285)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    3288..3309
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     promoter        3310..3344
                     /label=BBa_J23101
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.7"
     terminator      4102..4173
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4189..4216
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      4244..4301
                     /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        4397..4431
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     misc_RNA        complement(5955..6004)
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     terminator      complement(6073..6100)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(6116..6187)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ggtatcagct cactcaaagg cggtaatttg acagctagct cagtcctagg tataatgcta
       61 gcagttcgat gagagcgata acagacgaga aacgttccgt ccgtctgggt cagttgggca
      121 aagttgatga ccgggtcgtc cgttcctttt aagcgtagtt ttcgtcgttt gctgcaggcc
      181 ttttgtatag ttcatccatg ccatgtgtaa tcccagcagc tgttacaaac tcaagaagga
      241 ccatgtggtc tctcttttcg ttgggatctt tcgaaagggc agattgtgtg gacaggtaat
      301 ggttgtctgg taaaaggaca gggccatcgc caattggagt attttgttga taatggtctg
      361 ctagttgaac gcttccatct tcaatgttgt gtctaatttt gaagttaact ttgattccat
      421 tcttttgttt gtctgccatg atgtatacat tgtgtgagtt atagttgtat tccaacttgt
      481 gtccaagaat gtttccatct tctttaaaat caataccttt taactcgatt ctattaacaa
      541 gtgtatcacc ttcaaacttg acttcagcac gtgtcttata gttcccgtca tctttgaaaa
      601 atatagttct ttcctgtaca taaccttcgg gcatggcact cttgaaaaag tcatgccgtt
      661 tcatatgatc tgggtatctt gaaaagcatt gaacaccata agtcaaagta gtgacaagtg
      721 ttggccatgg aacaggtagt tttccagtag tgcaaataaa tttaagggta agttttccgt
      781 atgctgcatc accttcaccc tctccactga cagaaaattt gtgcccatta acattaccat
      841 ctaattcaac aagaattggg acaactccag tgaaaagttc ttctccttta ctcatctagt
      901 atttctcctc tttctctaga ttaaacaaaa ttatttgtag aggctgtttc gtcctcacgg
      961 actcatcaga ccggaaagca catccggtga cagctaagct gaacctgcgt aaaagttaag
     1021 gttaggcatg taaacttaac tctcacacag gtttataaca ccacagagga cacatgccct
     1081 acctccatga tctctagggc ggcggatttg tcctactcag gagagcgttc accgacaaac
     1141 aacagataaa acgaaaggcc cagtctttcg actgagcctt tcgttttatt tgatgccacg
     1201 cgtaccatgg gactagggcc catacccaaa aaaaaacccc gcccctgaca gggcggggtt
     1261 ttttttaggt agagacccac gaggcagacc tcagcgctag cggagtgtat actggcttac
     1321 tatgttggca ctgatgaggg tgtcagtgaa gtgcttcatg tggcaggaga aaaaaggctg
     1381 caccggtgcg tcagcagaat atgtgataca ggatatattc cgcttcctcg ctcactgact
     1441 cgctacgctc ggtcgttcga ctgcggcgag cggaaatggc ttacgaacgg ggcggagatt
     1501 tcctggaaga tgccaggaag atacttaaca gggaagtgag agggccgcgg caaagccgtt
     1561 tttccatagg ctccgccccc ctgacaagca tcacgaaatc tgacgctcaa atcagtggtg
     1621 gcgaaacccg acaggactat aaagatacca ggcgtttccc ctggcggctc cctcgtgcgc
     1681 tctcctgttc ctgcctttcg gtttaccggt gtcattccgc tgttatggcc gcgtttgtct
     1741 cattccacgc ctgacactca gttccgggta ggcagttcgc tccaagctgg actgtatgca
     1801 cgaacccccc gttcagtccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     1861 cccggaaaga catgcaaaag caccactggc agcagccact ggtaattgat ttagaggagt
     1921 tagtcttgaa gtcatgcgcc ggttaaggct aaactgaaag gacaagtttt ggtgactgcg
     1981 ctcctccaag ccagttacct cggttcaaag agttggtagc tcagagaacc ttcgaaaaac
     2041 cgccctgcaa ggcggttttt tcgttttcag agcaagagat tacgcgcaga ccaaaacgat
     2101 ctcaagaaga tcatcttatt aaggggtctg acgctcagtg gaacgaaaag tatatatgag
     2161 taaacttggt ctgacagctc gaggcttgga ttctcaccaa taaaaaacgc ccggcggcaa
     2221 ccgagcgttc tgaacaaatc cagatggagt tctgaggtca ttactggatc tatcaacagg
     2281 agtccaagcg agctcgatat caaattacgc cccgccctgc cactcatcgc agtactgttg
     2341 taattcatta agcattctgc cgacatggaa gccatcacaa acggcatgat gaacctgaat
     2401 cgccagcggc atcagcacct tgtcgccttg cgtataatat ttgcccatgg tgaaaacggg
     2461 ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac tcacccaggg
     2521 attggctgag acgaaaaaca tattctcaat aaacccttta gggaaatagg ccaggttttc
     2581 accgtaacac gccacatctt gcgaatatat gtgtagaaac tgccggaaat cgtcgtggta
     2641 ttcactccag agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt aacaagggtg
     2701 aacactatcc catatcacca gctcaccgtc tttcattgcc atacgaaatt ccggatgagc
     2761 attcatcagg cgggcaagaa tgtgaataaa ggccggataa aacttgtgct tatttttctt
     2821 tacggtcttt aaaaaggccg taatatccag ctgaacggtc tggttatagg tacattgagc
     2881 aactgactga aatgcctcaa aatgttcttt acgatgccat tgggatatat caacggtggt
     2941 atatccagtg atttttttct ccattttagc ttccttagct cctgaaaatc tcgataactc
     3001 aaaaaatacg cccggtagtg atcttatttc attatggtga aagttggaac ctcttacgtg
     3061 cccgatcaac tcgcgcgttt gccacctgac gtctaagaaa aggaatattc agcaatttgc
     3121 ccgtgccgaa gaaaggccca cccgtgaagg tgagccagtg agttgattgt gccacctgac
     3181 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggcag
     3241 aatttcagat aaaaaaaatc cttagctttc gctaaggatg atttctggaa ttcgcggccg
     3301 cttctagagt ttacagctag ctcagtccta ggtattatgc tagctactag agtcacacag
     3361 gaaagtacta gatggcttcc tccgaagacg ttatcaaaga gttcatgcgt ttcaaagttc
     3421 gtatggaagg ttccgttaac ggtcacgagt tcgaaatcga aggtgaaggt gaaggtcgtc
     3481 cgtacgaagg tacccagacc gctaaactga aagttaccaa aggtggtccg ctgccgttcg
     3541 cttgggacat cctgtccccg cagttccagt acggttccaa agcttacgtt aaacacccgg
     3601 ctgacatccc ggactacctg aaactgtcct tcccggaagg tttcaaatgg gaacgtgtta
     3661 tgaacttcga agacggtggt gttgttaccg ttacccagga ctcctccctg caagacggtg
     3721 agttcatcta caaagttaaa ctgcaagacg gtgagttcat ctacaaagtt aaactgcgtg
     3781 gtaccaactt cccgtccgac ggtccggtta tgcagaaaaa aaccatgggt tgggaagctt
     3841 ccaccgaacg tatgtacccg gaagacggtg ctctgaaagg tgaaatcaaa atgcgtctga
     3901 aactgaaaga cggtggtcac tacgacgctg aagttaaaac cacctacatg gctaaaaaac
     3961 cggttcagct gccgggtgct tacaaaaccg acatcaaact ggacatcacc tcccacaacg
     4021 aagactacac catcgttgaa cagtacgaac gtgctgaagg tcgtcactcc accggtgctt
     4081 aataatacta gagccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt
     4141 cgttttatct gttgtttgtc ggtgaacgct ctctactaga gtcacactgg ctcaccttcg
     4201 ggtgggcctt tctgcgttta tatactagta gcggcagctg cagtccggca aaaaagggca
     4261 aggtgtcacc accctgccct ttttctttaa aaccgaaaag attacttcgc gttatgcagg
     4321 cttcctcgct cactgactca ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     4381 actcaaaggc ggtaatttga cagctagctc agtcctaggt ataatgctag cagttcgatg
     4441 agagcgataa cgagcgccgg atcagggagt ggacgacctg ggagcgctac acgctgtggc
     4501 tgcggtcggt gcttatttca gggtgtaatc cagggtgatt tttttcagct tgcctttctc
     4561 gcgataaatc acaatctttt cgaagatgct gaacagcagc tcttttttgg taaagtcgtt
     4621 ggcctgctca aactgctgtt taaaatcgat cattttgctc aggcggttat atttctcttc
     4681 aaccggttct ttatcttctt ccacttcgct cagcttgcta tagatttctt cttctttttt
     4741 gttcagttcg ttaattttgg caaacagttc ttctttcgga atcgggttac gatcatccat
     4801 atacagatcg taccatttct ctttctggcg tttgattttt tccagttctt ttgtcagtgc
     4861 gctaacatca acaacctctt gctgatcata gcggtcgctg aatttgctca taaagtattc
     4921 tttgctggta atcagcagct gaatttcatc cagcagcggt tccagaatgt ttttttcgtt
     4981 ggtaatgcgg tgacatttgg tacaacggta ataggttttc tgctcacgtt tatcaaaatg
     5041 gccctgcatt ttatggccac cacaattacc acactgcagc agaccggtaa acagaaattt
     5101 accaacgcgt ttacgatcaa cgccacgttt ttctttttct ttctgggctt ttttgaacag
     5161 ggtttcgcta ataataggcg gatgattatt cggaacaact ttatcacccc aacgataggt
     5221 gccaatatag atatcgttgg tcaggatatc acgcacgcta ttatgattcc attctttggc
     5281 aatacgcggt ttaaccatca gttcattcag acgatctgca atgctacgat agccataacc
     5341 atcacaatac atgcgataga tcatacgaac aacgtctgct tcttcctcaa taatggtgca
     5401 attgaagtct ttatcaaatt tgtaaccata cgggctatga ccacccggtt ttttaccttc
     5461 atcgatcatc tgctcgatac caaatttaac acgttctgcc agattttcac gttcccactg
     5521 tgccagtgct gcaaccaggg taataaacag acgacccatt gcggtgctgg tatcataaac
     5581 ttcggtggcg ctacgaaatg ccacattgta tttttcaaag gtctgcagca gcagatacag
     5641 atccagaacg ctacgggtca ggcgatccag acgatacacc agcacaatat caatgttgcc
     5701 ttttttgata tctttcagca gacgctgcat ctgcggacga tccagatctt ttgcgctcca
     5761 accttcttca atatattctt gcacaatttc ccaaccctgg cttgcacaaa atgcacgcag
     5821 acgttcacgc tgtgccggaa tgctaaaacc ttctttggcc tgttcatcgg tgctaacacg
     5881 aacataaatg gccactttca ttcgaatcct agttattttg ttgaaattac tttaaacaaa
     5941 attatttgta gaggctgttt cgtcctcacg gactcatcag accggaaagc acatccggtg
     6001 acagctcaga tcagggtgcg caagttgtca acgctcccag gagagttatc gacttgcgta
     6061 ttagggtata aacgcagaaa ggcccacccg aaggtgagcc agtgtgactc tagtagagag
     6121 cgttcaccga caaacaacag ataaaacgaa aggcccagtc tttcgactga gcctttcgtt
     6181 ttatttgatg cctggctagg gcccataccc aaaaaaaaac cccgcccctg acagggcggg
     6241 gtttttttta ggtagagacc cacgaggcag acctca
//
