LOCUS       sequence-216433-        4968 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For one-step cloning (with blue/ white screening) of an open reading
            frame of interest for constitutive gene expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4968)
  TITLE     For one step cloning of various constructs including blue/ white 
            screening
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4968)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pCMV6-LacZ-BbsI
FEATURES             Location/Qualifiers
     source          1..4968
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     166..182
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        343..722
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        723..926
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     877..897
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        952..970
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1017..1038
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1053..1058,1059..1076,1077..1083)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1091..1107
                     /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     1096..1118
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1115..1131
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1115..1131
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1127..1405
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEW"
     primer_bind     1150..1170
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1152..1170
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(1218..1236)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1246..1262)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1451..1470)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     rep_origin      complement(2023..2611)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2103..2122)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    2940..2987
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     3012..3031
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(3219..4013)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(3219..4013)
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3330..3349)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3940..3959
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4048..4405)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4062..4197
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        4214..4405
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     4362..4382
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(4407..4511)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4538..4966
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4625..4644)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4835..4856
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 aacaaaatat taacgcttac aatttccatt cgccattcag gctgcgcaac tgttgggaag
       61 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
      121 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
      181 gtgccaagct gatctataca ttgaatcaat attggcaatt agccatatta gtcattggtt
      241 atatagcata aatcaatatt ggctattggc cattgcatac gttgtatcta tatcataata
      301 tgtacattta tattggctca tgtccaatat gaccgccatg ttgacattga ttattgacta
      361 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
      421 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
      481 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
      541 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
      601 gtccgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
      661 tgaccttacg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
      721 tggtgatgcg gttttggcag tacaccaatg ggcgtggata gcggtttgac tcacggggat
      781 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
      841 actttccaaa atgtcgtaat aaccccgccc cgttgacgca aatgggcggt aggcgtgtac
      901 ggtgggaggt ctatataagc agagctcgtt tagtgaaccg tcagaatttt gtaatacgac
      961 tcactatagg gcggccgggt cttcatctgg aaagcgggca gtgagcgcaa cgcaattaat
     1021 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
     1081 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
     1141 gccaagcgcg caattaaccc tcactaaagg gaacaaaagc tggaggacct cgaggggggg
     1201 cccggtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt
     1261 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     1321 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     1381 gcgcagcctg aatggcgaat ggtaggacgc gccctgtagc gtgggaagac aacctgtagg
     1441 gcctgcgggg tctattggga accaagctgg agtgcagtgg cacaatcttg gctcactgca
     1501 atctccgcct cctgggttca agcgattctc ctgcctcagc ctcccgagtt gttgggattc
     1561 caggcatgca tgaccaggct cagctaattt ttgttttttt ggtagagacg gggtttcacc
     1621 atattggcca ggctggtctc caactcctaa tctcaggtga tctacccacc ttggcctccc
     1681 aaattgctgg gattacaggc gtgaaccact gctcccttcc ctgtccttct gattttaaaa
     1741 taactatacc agcaggagga cgtccagaca cagcataggc tacctggcca tgcccaaccg
     1801 gtgggacatt tgagttgctt gcttggcact gtcctctcat gcgttgggtc cactcagtag
     1861 atgcctgttg aattgggtac gcggccagcg gcgagcggta tcagctcact caaaggcggt
     1921 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     1981 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     2041 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     2101 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     2161 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     2221 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     2281 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     2341 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     2401 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     2461 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     2521 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     2581 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     2641 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     2701 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     2761 tgagtaacct gaggctatgg cagggcctgc cgccccgacg ttggctgcga gccctgggcc
     2821 ttcacccgaa cttggggggt ggggtgggga aaaggaagaa acgcgggcgt attggcccca
     2881 atggggtctc ggtggggtat cgacagagtg ccagccctgg gaccgaaccc cgcgtttatg
     2941 aacaaacgac ccaacaccgt gcgttttatt ctgtcttttt attgccgtca tagcgcgggt
     3001 tccttccggt attgtctcct tccgtgtttc agttagcctc cccctagggt gggcgaagaa
     3061 ctccagcatg agatccccgc gctggaggat catccagccg gcgtcccgga aaacgattcc
     3121 gaagcccaac ctttcataga aggcggcggt ggaatcgaaa tctcgtgatg gcaggttggg
     3181 cgtcgcttgg tcggtcattt cgaaccccag agtcccgctc agaagaactc gtcaagaagg
     3241 cgatagaagg cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg
     3301 tcagcccatt cgccgccaag ctcttcagca atatcacggg tagccaacgc tatgtcctga
     3361 tagcgatccg ccacacccag ccggccacag tcgatgaatc cagaaaagcg gccattttcc
     3421 accatgatat tcggcaagca ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc
     3481 atgctcgcct tgagcctggc gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc
     3541 agatcatcct gatcgacaag accggcttcc atccgagtac gtgctcgctc gatgcgatgt
     3601 ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca
     3661 tcagccatga tggatacttt ctcggcagga gcaaggtgag atgacaggag atcctgcccc
     3721 ggcacttcgc ccaatagcag ccagtccctt cccgcttcag tgacaacgtc gagcacagct
     3781 gcgcaaggaa cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc ttgcagttca
     3841 ttcagggcac cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc
     3901 cggaacacgg cggcatcaga gcagccgatt gtctgttgtg cccagtcata gccgaatagc
     3961 ctctccaccc aagcggccgg agaacctgcg tgcaatccat cttgttcaat catgcgaaac
     4021 gatcctcatc ctgtctcttg atcgatcttt gcaaaagcct aggcctccaa aaaagcctcc
     4081 tcactacttc tggaatagct cagaggccga ggcggcctcg gcctctgcat aaataaaaaa
     4141 aattagtcag ccatggggcg gagaatgggc ggaactgggc ggagttaggg gcgggatggg
     4201 cggagttagg ggcgggacta tggttgctga ctaattgaga tgcatgcttt gcatacttct
     4261 gcctgctggg gagcctgggg actttccaca cctggttgct gactaattga gatgcatgct
     4321 ttgcatactt ctgcctgctg gggagcctgg ggactttcca caccctaact gacacacatt
     4381 ccacagctgg ttctttccgc ctcaggactc ttcctttttc aatattattg aagcatttat
     4441 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     4501 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg cgccctgtag cggcgcatta
     4561 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     4621 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     4681 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     4741 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     4801 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     4861 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     4921 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaatttt
//
