LOCUS       sequence-163261-        6939 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pCALNL reporter contains two target sites consisting of PAM Cas9 
            site-gix psuedo site-Cas9 site-PAM that match region in centromeres 
            of chromosomes 5 (numbers in common name refer to pos. in hg38).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6939)
  AUTHORS   Chaikind B, Bessen JL, Thompson DB, Hu JH, Liu DR
  TITLE     A programmable Cas9-serine recombinase fusion protein that operates 
            on DNA sequences in mammalian cells.
  JOURNAL   Nucleic Acids Res. 2016 Aug 11. pii: gkw707.
  PUBMED    27515511
REFERENCE   2  (bases 1 to 6939)
  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: "Nucleic 
            Acids Res. 2016 Aug 11. pii: gkw707."
COMMENT     Sequence Label: pCALNL_Ch5_155183064-155183141
FEATURES             Location/Qualifiers
     source          1..6939
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1674
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1598..1620
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1682..1701
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1877..2671
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSSQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1931..1950)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2541..2560
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2851..2985
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     regulatory      3149..3158
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3155..3157,3158..3160,3161..3874)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3155..3157,3158..3160,3161..3871)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3200..3221)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3461..3480)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3808..3829
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3961..3980)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4026..4081
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4080..4099)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4442..4458)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4442..4458)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4455..4477)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4466..4482
                     /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(4490..4496,4497..4514,4515..4520))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4535..4556
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4615..4810
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4661..4796
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4816..4950
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5018..5035)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5189..5777)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5269..5288)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5948..6739,6740..6808))
                     /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     6571..6590
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6809..6913)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc
     1321 tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag
     1381 ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc
     1441 tctagcgggc gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc
     1501 cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg
     1561 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
     1621 ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg
     1681 ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattgat taattcgagc
     1741 gaacgcgtcc caccggctca tgagaggtag agctaaggtc caaacctagg tttatctgag
     1801 accggaactc atgtgattaa ctgtggagtc gagtcggatt tgatctgatc aagagacagg
     1861 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     1921 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     1981 cgtgttccgg ctgtcatcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     2041 tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     2101 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     2161 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     2221 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     2281 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     2341 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     2401 ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     2461 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     2521 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     2581 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     2641 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     2701 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     2761 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     2821 atgctggagt tcttcgccca ccccatcgat aacttgttta ttgcagctta taatggttac
     2881 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     2941 tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatcaaatc cgaacgcacc
     3001 ccaccggctc atgagaggta gagctaaggt ccaaacctag gtttatctga gaccggaact
     3061 catgtgatta actgtggctc gagtcgctcg gtacgattta aattgaattc tgcagtcgac
     3121 ggtaccgcgg gcccgggatc caccggtcgc caccatggtg agcaagggcg aggagctgtt
     3181 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     3241 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     3301 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
     3361 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     3421 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     3481 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     3541 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     3601 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     3661 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     3721 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag
     3781 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     3841 gatcactctc ggcatggacg agctgtacaa gtaaagcggc cgcactcctc aggtgcaggc
     3901 tgcctatcag aaggtggtgg ctggtgtggc caatgccctg gctcacaaat accactgaga
     3961 tctttttccc tctgccaaaa attatgggga catcatgaag ccccttgagc atctgacttc
     4021 tggctaataa aggaaattta ttttcattgc aatagtgtgt tggaattttt tgtgtctctc
     4081 actcggaagg acatatggga gggcaaatca tttaaaacat cagaatgagt atttggttta
     4141 gagtttggca acatatgccc atatgctggc tgccatgaac aaaggttggc tataaagagg
     4201 tcatcagtat atgaaacagc cccctgctgt ccattcctta ttccatagaa aagccttgac
     4261 ttgaggttag atttttttta tattttgttt tgtgttattt ttttctttaa catccctaaa
     4321 attttcctta catgttttac tagccagatt tttcctcctc tcctgactac tcccagtcat
     4381 agctgtccct cttctcttat gaagatccct cgacctgcag cccaagcttg gcgtaatcat
     4441 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     4501 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     4561 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagcgg atccgcatct
     4621 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     4681 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     4741 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     4801 cttttgcaaa aagctaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     4861 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     4921 ctcatcaatg tatcttatca tgtctggatc cgctgcatta atgaatcggc caacgcgcgg
     4981 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     5041 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     5101 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     5161 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     5221 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     5281 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     5341 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt
     5401 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     5461 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     5521 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     5581 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     5641 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     5701 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     5761 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     5821 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     5881 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     5941 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     6001 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     6061 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     6121 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     6181 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     6241 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     6301 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     6361 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     6421 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     6481 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     6541 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     6601 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     6661 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     6721 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     6781 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     6841 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     6901 taggggttcc gcgcacattt ccccgaaaag tgccacctg
//
