LOCUS       sequence-163268-        6937 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 13 (numbers in common name refers to pos. in hg38).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6937)
  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 6937)
  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_chr13_102010574-102010650
FEATURES             Location/Qualifiers
     source          1..6937
                     /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             1876..2670
                     /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(1930..1949)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2540..2559
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2850..2984
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     regulatory      3147..3156
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3153..3155,3156..3158,3159..3872)
                     /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(3153..3155,3156..3158,3159..3869)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3198..3219)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3459..3478)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3806..3827
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3959..3978)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4024..4079
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4078..4097)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4440..4456)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4440..4456)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4453..4475)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4464..4480
                     /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(4488..4494,4495..4512,4513..4518))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4533..4554
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4613..4808
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4659..4794
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4814..4948
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5016..5033)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5187..5775)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5267..5286)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5946..6737,6738..6806))
                     /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     6569..6588
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6807..6911)
                     /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 tagggaagtg atcatagctg agtttctgga aaaacctagg ttttaaagtt
     1801 gaggagactt aagtccaaaa cctggagtcg agtcggattt gatctgatca agagacagga
     1861 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     1921 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     1981 gtgttccggc tgtcatcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     2041 gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     2101 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     2161 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     2221 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     2281 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     2341 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     2401 gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     2461 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     2521 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     2581 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     2641 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     2701 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     2761 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     2821 tgctggagtt cttcgcccac cccatcgata acttgtttat tgcagcttat aatggttaca
     2881 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     2941 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatcaaatcc gaacgcaccc
     3001 tagggaagtg atcatagctg agtttctgga aaaacctagg ttttaaagtt gaggagactt
     3061 aagtccaaaa cctggctcga gtcgctcggt acgatttaaa ttgaattctg cagtcgacgg
     3121 taccgcgggc ccgggatcca ccggtcgcca ccatggtgag caagggcgag gagctgttca
     3181 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     3241 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
     3301 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc
     3361 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     3421 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     3481 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     3541 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
     3601 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc
     3661 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     3721 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca
     3781 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     3841 tcactctcgg catggacgag ctgtacaagt aaagcggccg cactcctcag gtgcaggctg
     3901 cctatcagaa ggtggtggct ggtgtggcca atgccctggc tcacaaatac cactgagatc
     3961 tttttccctc tgccaaaaat tatggggaca tcatgaagcc ccttgagcat ctgacttctg
     4021 gctaataaag gaaatttatt ttcattgcaa tagtgtgttg gaattttttg tgtctctcac
     4081 tcggaaggac atatgggagg gcaaatcatt taaaacatca gaatgagtat ttggtttaga
     4141 gtttggcaac atatgcccat atgctggctg ccatgaacaa aggttggcta taaagaggtc
     4201 atcagtatat gaaacagccc cctgctgtcc attccttatt ccatagaaaa gccttgactt
     4261 gaggttagat tttttttata ttttgttttg tgttattttt ttctttaaca tccctaaaat
     4321 tttccttaca tgttttacta gccagatttt tcctcctctc ctgactactc ccagtcatag
     4381 ctgtccctct tctcttatga agatccctcg acctgcagcc caagcttggc gtaatcatgg
     4441 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     4501 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     4561 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagcggat ccgcatctca
     4621 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     4681 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     4741 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     4801 tttgcaaaaa gctaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     4861 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     4921 catcaatgta tcttatcatg tctggatccg ctgcattaat gaatcggcca acgcgcgggg
     4981 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     5041 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     5101 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     5161 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     5221 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     5281 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5341 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat
     5401 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5461 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5521 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5581 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
     5641 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5701 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5761 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5821 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5881 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5941 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     6001 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     6061 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     6121 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     6181 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     6241 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6301 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     6361 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6421 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6481 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6541 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6601 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6661 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6721 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6781 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6841 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6901 ggggttccgc gcacatttcc ccgaaaagtg ccacctg
//
