LOCUS       sequence-454041-        6429 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of human codon-optimized Bxb1 mutant N194G.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6429)
  AUTHORS   Jiang K, Yan Z, Di Bernardo M, Sgrizzi SR, Villiger L, Kayabolen A, 
            Kim B, Carscadden JK, Hiraizumi M, Nishimasu H, Gootenberg JS, 
            Abudayyeh OO
  TITLE     Rapid in silico directed evolution by a protein language model with 
            EVOLVEpro
  JOURNAL   Science 0, eadr6006
  PUBMED    39071429
REFERENCE   2  (bases 1 to 6429)
  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: "Science 0, 
            eadr6006"
COMMENT     Sequence Label: pKJ0376
FEATURES             Location/Qualifiers
     source          1..6429
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    16..37
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(52..57,58..75,76..82)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    90..106
                     /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)."
     primer_bind     95..117
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     114..130
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     114..130
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        150..529
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          852..1868
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1792..1814
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1881..1900
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1881..1899
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1919..1939
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1940..1966
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(3492..3509)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3498..3722
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3794..3811)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3794..3810)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3803..3825)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(3951..4406)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4088..4109)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4300..4319
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4433..4537
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4538..4606,4607..5398)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4756..4775)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5569..6157
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6058..6077
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6311..6328
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
       61 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
      121 cagctatgac catgaggcgc gccggattcg acattgatta ttgactagtt attaatagta
      181 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
      241 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
      301 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
      361 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
      421 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
      481 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tcgaggtgag
      541 ccccacgttc tgcttcactc tccccatctc ccccccctcc ccacccccaa ttttgtattt
      601 atttattttt taattatttt gtgcagcgat gggggcgggg gggggggggg gggcgcgccg
      661 gggggggggg gggggggggg gggggggggg gggggcggga gggggggggg gggggggggg
      721 gggggggggg ggggggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc
      781 tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg
      841 cgcggcgggc gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct
      901 cgcgccgccc gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg
      961 gcccttctcc tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt
     1021 ggctgcgtga aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg
     1081 ggggtgcgtg cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg
     1141 gctgtgagcg ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga
     1201 gcgcggccgg gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg
     1261 tgcggggtgt gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac
     1321 cccccctgca cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc
     1381 gtacggggcg tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc
     1441 cgggcggggc ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc
     1501 ggagcgccgg cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt
     1561 gcgagagggc gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc
     1621 gccgccgcac cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa
     1681 tgggcgggga gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc
     1741 ggggctgtcc gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct
     1801 tctggcgtgt gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt
     1861 tcctacagat ccttaattaa taatacgact cactataggg ggtcgacccg ccaccatgcc
     1921 aaaaaagaaa agaaaagtgt atccctatga tgtccccgat tatgccggtt caagagccct
     1981 ggtcgtgatt agactgagcc gagtgacaga cgccaccaca agtcccgaga gacagctgga
     2041 atcatgccag cagctctgtg ctcagcgggg ttgggatgtg gtcggcgtgg cagaggatct
     2101 ggacgtgagc ggggccgtcg atccattcga cagaaagagg aggcccaacc tggcaagatg
     2161 gctcgctttc gaggaacagc cctttgatgt gatcgtcgcc tacagagtgg accggctgac
     2221 ccgctcaatt cgacatctcc agcagctggt gcattgggct gaggaccaca agaaactggt
     2281 ggtcagcgca acagaagccc acttcgatac taccacacct tttgccgctg tggtcatcgc
     2341 actgatgggc actgtggccc agatggagct cgaagctatc aaggagcgaa acaggagcgc
     2401 agcccatttc aatattaggg ccggtaaata cagaggctcc ctgccccctt ggggatatct
     2461 ccctaccagg gtggatgggg agtggagact ggtgccagac cccgtccaga gagagcggat
     2521 tctggaagtg taccacagag tggtcgatgg tcacgaacca ctccatctgg tggcacacga
     2581 cctgaataga cgcggcgtgc tctctccaaa ggattatttt gctcagctgc agggaagaga
     2641 gccacaggga agagaatgga gtgctactgc actgaagaga tctatgatca gtgaggctat
     2701 gctgggttac gcaacactca atggcaaaac tgtccgggac gatgacggag cccctctggt
     2761 gagggctgag cctattctca ccagagagca gctcgaagct ctgcgggcag aactggtcaa
     2821 gactagtcgc gccaaacctg ccgtgagcac cccaagcctg ctcctgaggg tgctgttctg
     2881 cgccgtctgt ggagagccag catacaagtt tgccggcgga gggcgcaaac atccccgcta
     2941 tcgatgcagg agcatggggt tccctaagca ctgtggaaac gggacagtgg ccatggctga
     3001 gtgggacgcc ttttgcgagg aacaggtgct ggatctcctg ggtgacgctg agcggctgga
     3061 aaaagtgtgg gtggcaggat ctgactccgc tgtggagctg gcagaagtca atgccgagct
     3121 cgtggatctg acttccctca tcggatctcc tgcatataga gctgggtccc cacagagaga
     3181 agctctggac gcacgaattg ctgcactcgc tgctagacag gaggaactgg agggcctgga
     3241 ggccaggccc tctggatggg agtggcgaga aaccggacag aggtttgggg attggtggag
     3301 ggagcaggac accgcagcca agaacacatg gctgagatcc atgaatgtcc ggctcacatt
     3361 cgacgtgcgc ggtggcctga ctcgaaccat cgattttggc gacctgcagg agtatgaaca
     3421 gcacctgaga ctggggtccg tggtcgaaag actgcacact gggatgtcct aggtcagagc
     3481 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     3541 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     3601 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     3661 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     3721 ggcttctgag gcggaaagaa ccagctgggg ctcgagatcc actagttcta gcctcgaggc
     3781 tagagcggcc gccactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
     3841 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
     3901 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg aaattgtaag
     3961 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     4021 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     4081 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     4141 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     4201 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     4261 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     4321 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     4381 gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac
     4441 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     4501 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     4561 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     4621 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     4681 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     4741 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     4801 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     4861 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     4921 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     4981 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     5041 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     5101 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     5161 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     5221 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     5281 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     5341 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     5401 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     5461 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     5521 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     5581 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     5641 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     5701 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     5761 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     5821 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     5881 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     5941 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     6001 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     6061 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     6121 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     6181 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     6241 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     6301 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
     6361 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     6421 agcgggcag
//
