LOCUS       sequence-354284-        6638 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PhiBT Integrase expression vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6638)
  AUTHORS   Yarnall MTN, Ioannidi EI, Schmitt-Ulms C, Krajeski RN, Lim J, 
            Villiger L, Zhou W, Jiang K, Garushyants SK, Roberts N, Zhang L, 
            Vakulskas CA, Walker JA 2nd, Kadina AP, Zepeda AE, Holden K, Ma H, 
            Xie J, Gao G, Foquet L, Bial G, Donnelly SK, Miyata Y, Radiloff DR, 
            Henderson JM, Ujita A, Abudayyeh OO, Gootenberg JS
  TITLE     Drag-and-drop genome insertion of large sequences without 
            double-strand DNA cleavage using CRISPR-directed integrases.
  JOURNAL   Nat Biotechnol. 2022 Nov 24. doi: 10.1038/s41587-022-01527-4.
  PUBMED    36424489
REFERENCE   2  (bases 1 to 6638)
  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: "Nat 
            Biotechnol. 2022 Nov 24. doi: 10.1038/s41587-022-01527-4."
COMMENT     Sequence Label: pDY0312 PhiBT Integrase
FEATURES             Location/Qualifiers
     source          1..6638
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        6..385
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        387..664
                     /label=chicken beta-actin promoter
     intron          665..1681
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1605..1627
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1694..1713
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1694..1712
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1732..1752
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3557..3574)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3563..3787
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3859..3876)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3859..3875)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3868..3890)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(4016..4471)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4153..4174)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4365..4384
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4498..4602
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4603..4671,4672..5463)
                     /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(4821..4840)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5634..6222
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6123..6142
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6376..6393
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6510..6531
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6546..6551,6552..6569,6570..6576)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6584..6600
                     /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     6589..6611
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6608..6624
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6608..6624
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gattcgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca
       61 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
      121 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
      181 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
      241 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
      301 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
      361 cgtattagtc atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc
      421 catctccccc ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc
      481 agcgatgggg gcgggggggg ggggggggcg cgcgccrggs ggggsggggs ggggsgrggg
      541 gsggggsggg gsgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa
      601 gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg
      661 ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg
      721 cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc
      781 tcctccgggc tgtaattagc gcttggttta atgacggctt gtttcttttc tgtggctgcg
      841 tgaaagcctt gaggggctcc gggagggccc tttgtgcggg gggagcggct cggggggtgc
      901 gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc gcgctgcccg gcggctgtga
      961 gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt gtgcgcgagg ggagcgcggc
     1021 cgggggcggt gccccgcggt gcgggggggg ctgcgagggg aacaaaggct gcgtgcgggg
     1081 tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg gtcgggctgc aaccccccct
     1141 gcacccccct ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc tccgtacggg
     1201 gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg
     1261 ggcggggccg cctcgggccg gggagggctc gggggagggg cgcggcggcc cccggagcgc
     1321 cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat cgtgcgagag
     1381 ggcgcaggga cttcctttgt cccaaatctg tgcggagccg aaatctggga ggcgccgccg
     1441 caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg
     1501 ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc ctcggggctg
     1561 tccgcggggg gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg
     1621 tgtgaccggc ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca
     1681 gatccttaat taataatacg actcactata gggggtcgac ccgccaccat gcccaagaag
     1741 aaacggaaag tgatgagccc ctttatcgcc ccggacgtgc ccgagcacct cctggacact
     1801 gtgcgcgtct ttctgtacgc ccgtcagagt aaaggacggt cagatggatc tgacgtgtcc
     1861 accgaagcac agctcgctgc cggacgggcc cttgttgcct caagaaacgc acaaggggga
     1921 gctagatggg tggtggcggg cgaattcgtg gatgtgggca gatcagggtg ggacccgaat
     1981 gtgacacgcg ccgacttcga aagaatgatg ggcgaggtgc gcgccggtga gggagacgta
     2041 gtggtggtta atgaactgag tcgccttacg aggaagggcg cccacgacgc tctggagatc
     2101 gataacgaac tcaaaaaaca cggtgtgcgg ttcatgagcg tgctggaacc attcctggat
     2161 accagcaccc caatcggtgt cgcgatcttt gccctgattg ccgcgctcgc taaacaggat
     2221 tcagacctta aagctgagcg gctgaagggg gctaaagatg agatcgctgc cttggggggt
     2281 gtgcacagct catctgcgcc attcggcatg agggcggtca gaaagaaagt ggataacctg
     2341 gtcatatctg ttctggagcc tgatgaggac aacccggacc acgttgagct tgtggaacgg
     2401 atggctaaga tgtctttcga aggcgtcagc gataacgcaa ttgccacaac atttgagaag
     2461 gagaaaatcc cctctccggg gatggctgag agacgagcca cggagaagag gcttgcttct
     2521 attaaggcac ggaggctcaa tggcgccgaa aagccgatca tgtggcgggc gcagacagtt
     2581 agatggattc ttaaccatcc cgcgattggt ggattcgcat tcgagcgggt gaaacacgga
     2641 aaagcccaca tcaacgtgat acgaagagat cccggcggca aaccccttac ccctcacact
     2701 ggtatcctgt ctggatccaa gtggttggaa ctccaggaga agagaagcgg gaaaaatctc
     2761 tccgaccgca aaccaggtgc cgaagtggaa cctacgctgc tttccgggtg gagatttctg
     2821 ggatgtcgga tatgcggtgg gtcaatgggc cagtcccaag ggggccgtaa gaggaatggg
     2881 gacttggctg agggcaatta catgtgtgca aacccaaagg ggcacggcgg tctgagcgtc
     2941 aagaggtctg agcttgatga attcgtggca tcaaaagtct gggccaggtt gcgcacggct
     3001 gacatggagg atgaacatga ccaagcatgg attgcagctg cagctgaacg gtttgctttg
     3061 cagcacgacc tggcgggggt agctgacgag cgacgggagc aacaagctca cctggataac
     3121 gttcggagat caataaaaga tctccaggcg gataggaagg caggtctcta cgtgggacgc
     3181 gaagaactgg agacctggcg cagtaccgtc ctgcaatata ggagctacga ggctgagtgt
     3241 actactaggt tggctgagct ggatgaaaaa atgaatggat ccacccgggt gccttcagaa
     3301 tggtttagcg gcgaggaccc aaccgcggaa ggaggcatat gggcgagctg ggatgtctat
     3361 gagcgccggg agtttctcag cttttttttg gactccgtaa tggttgacag gggcagacat
     3421 cctgaaacca agaaatatat accattgaaa gaccgggtga ccttaaagtg ggcggagctg
     3481 ttaaaggaag aggatgaagc aagcgaggcc acagaacggg agctggcagc tctttaggtc
     3541 agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     3601 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     3661 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     3721 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     3781 tctatggctt ctgaggcgga aagaaccagc tggggctcga gatccactag ttctagcctc
     3841 gaggctagag cggccgccac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     3901 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
     3961 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggaaatt
     4021 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     4081 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     4141 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     4201 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     4261 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     4321 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     4381 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     4441 gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc
     4501 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     4561 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     4621 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     4681 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     4741 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     4801 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     4861 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     4921 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     4981 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     5041 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     5101 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     5161 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     5221 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     5281 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     5341 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     5401 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     5461 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     5521 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     5581 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     5641 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     5701 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     5761 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     5821 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     5881 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     5941 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     6001 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     6061 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     6121 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     6181 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     6241 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     6301 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     6361 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
     6421 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     6481 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
     6541 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
     6601 tttcacacag gaaacagcta tgaccatgag gcgcgccg
//
