LOCUS       sequence-320248-        4738 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of ZF21i under the CMV promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4738)
  TITLE     Leonard Lab COMET plasmids
REFERENCE   2  (bases 1 to 4738)
  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"
COMMENT     Sequence Label: pPD688 ZF21i
FEATURES             Location/Qualifiers
     source          1..4738
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             910..975
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             982..1002
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(1368..1385)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1374..1598
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1644..2072
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1731..1750)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1941..1962
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2095..2291
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2142..2277
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2204..2223
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    2411..2532
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2448..2467)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2502..2521
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2581..2597)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2581..2597)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2594..2616)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2605..2621
                     /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(2629..2635,2636..2653,2654..2659))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2674..2695
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2812..2829)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2983..3571)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3063..3082)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3742..4533,4534..4602))
                     /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     4365..4384
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4603..4707)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagtcccaa gctggctagc
      901 gccaccatgg attataagga tcacgatggc gactacaagg accacgatat tgactacaaa
      961 gacgatgacg ataaaatggc tcctaagaaa aagcgcaaag tcggtatcca tggcgttccc
     1021 ggatcctctg gctcttcagg taccggtgga gctagacccg gggagcgccc cttccagtgt
     1081 cgcatttgca tgcggaactt ttcgagaaat ttcattttgc aaagacatac ccgtactcat
     1141 accggtgaaa aaccgtttca gtgtcggatc tgtatgcgaa atttctccca aggtggtaat
     1201 ttggttagac atctacgtac gcacaccggc gagaagccat tccaatgccg aatatgcatg
     1261 cgcaacttca gtcaacaaac tggtttgaat gttcacctaa aaacccacct gagaggatct
     1321 taagcggccg ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc
     1381 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     1441 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     1501 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaagt
     1561 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     1621 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     1681 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     1741 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     1801 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     1861 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     1921 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     1981 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     2041 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggc
     2101 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     2161 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     2221 aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     2281 gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggaggttgg gcttcggaat
     2341 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     2401 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     2461 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     2521 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     2581 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     2641 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     2701 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     2761 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     2821 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     2881 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     2941 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     3001 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     3061 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     3121 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     3181 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     3241 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     3301 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     3361 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     3421 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     3481 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     3541 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     3601 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     3661 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     3721 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     3781 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     3841 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagatccac gctcaccggc
     3901 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     3961 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     4021 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     4081 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     4141 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     4201 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     4261 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     4321 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     4381 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     4441 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     4501 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     4561 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     4621 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     4681 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
